Antibodies that bind psma and gamma-delta t cell receptors

ABSTRACT

Provided herein are antibodies capable of binding human PSMA and capable of binding a human Vγ9Vδ2 T cell receptor. In particular, provided herein are pharmaceutical compositions comprising the antibodies capable of binding human PSMA and capable of binding a human Vγ9VΩ T cell receptor and uses of the antibodies for medical treatment.

FIELD OF THE INVENTION

The present invention relates to novel multispecific antibodies capableof binding human PSMA and capable of binding a human Vγ9Vβ2 T cellreceptor. The invention further relates to pharmaceutical compositionscomprising the antibodies of the invention and to uses of the antibodiesof the invention for medical treatment.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common cancer in men in Europe and theUnited States. Early detection of localized disease results in highsurvival rates. However, metastasized tumors lead to dramaticallyreduced survival. Prostate-specific membrane antigen (PSMA), also knownas folate hydrolase I or glutamate carboxypeptidase II, is a potentialtarget for drug development. PSMA is a type II transmembrane proteinshowing overexpression on prostatic cancer cells, but low expression innormal tissues.

Several PSMA-targeting molecules have been developed (see e.g. Haberkornet a. (2016) Clin Cancer Res 22:9), including antibodies (Chatalic etal. (2015) J Nucl Med 56:1094; WO2018098354). Bispecific PSMA+CD3 T-cellengaging antibody approaches have also been described (Hernandez-Hoyoset al. (2016) Mol Cancer Ther 15:2155; WO2016187594). Bispecific T-cellengaging antibodies have a tumor target binding specificity and a T-cellbinding specificity and thus boost efficacy by redirecting T-cellcytotoxicity to malignant cells, see e.g. Huehls et al. (2015) ImmunolCell Biol 93:290; Ellerman (2019) Methods, 154:102; de Bruin et al.(2017) Oncoimmunology 7(1):e1375641 and WO2015156673. However, resultsvary significantly. For example, in one study in which a CD3 bindingmoiety was combined with binding moieties against 8 different B-celltargets (CD20, CD22, CD24, CD37, CD70, CD79b, CD138 and HLA-DR), it wasfound that the bispecific antibodies targeting the different tumortargets showed strong variation in their capacity to induce target cellcytotoxicity and that cytotoxicity did not correlate with antigenexpression levels. For example, CD3-based bispecific antibodiestargeting HLA-DR or CD138 were not able to induce cytotoxicity in spiteof intermediate to high HLA-DR and CD138 expression levels (Engelbertset al. (2020) Ebiomedicine 52:102625). Few T-cell redirecting therapieshave reached late-stage clinical development, possibly due tosignificant toxicity, manufacturing problems, immunogenicity, and lowresponse rates in solid tumors. In particular, toxicity may occur whenthe T-cell engager includes a CD3 binding arm as a result ofuncontrolled immune activation and cytokine release.

Thus, while significant progress has been made, there is still a needfor novel PSMA antibodies that are therapeutically effective yet haveacceptable toxicity. Such novel PSMA antibodies should also haveappropriate pharmacokinetic and pharmacodynamic properties and optimallybe manufacturable with high yield and purity. Furthermore, stableformations of such antibodies are needed in which minimal degradationand aggregation occurs.

These needs are addressed by the present invention.

SUMMARY OF THE INVENTION

The present invention provides novel antibodies for PSMA-based therapy.Bispecific antibodies were constructed in which PSMA-binding regionswere combined with binding regions capable of binding a Vγ9Vβ2 T cellreceptor and thus engaging γδ T cells. Surprisingly, the bispecificantibodies were able to mediate activation of autologous Vγ9Vδ2 T cells,including tumor-infiltrating Vγ9Vδ2 T cells, and to induce killing ofpatient-derived tumor cells in the presence of autologous PBMC-derivedVγ9Vδ2 T cells with very high potency. These activities were alsoobserved using tumor cell lines at low effector cell (γδ T cell) totarget cell (tumor cell) ratios, which is important, because γδ T cellsare only a subpopulation of T cells in humans which can vary in numbers.Normal healthy tissue, on the other hand, was not affected, indicatingthe potential of these antibodies for an efficacious yet safe cancertreatment. In addition, studies with whole human blood indicate that inspite of the high potency on target cells, the antibody only induced lowlevels of cytokine release, suggesting a low risk of cytokine releasesyndrome.

Furthermore, a novel bispecific VHH-human-Fc-containing antibody formatwas developed which, when produced in mammalian host cells, yielded ahighly homogenous and pure product. Moreover, the format had suitablepharmacokinetic properties for use in medical treatment and a suitablestable formulation for this product was developed. Without being boundby any specific theory, the molecular size and decreased hydrodynamicradius of the format may be highly suitable for tumor penetration, yetavoid renal filtration and ensure, via binding to FcRn, protection fromdegradation.

Accordingly, in a first aspect, the invention provides a multispecificantibody comprising a first antigen-binding region capable of bindinghuman PSMA and a second antigen-binding region capable of binding ahuman Vγ9Vβ2 T cell receptor.

In further aspects, the invention relates to pharmaceutical compositionscomprising the antibodies of the invention, uses of the antibodies ofthe invention in medical treatment, and to nucleic acid constructs,expression vectors for producing antibodies of the invention and to hostcells comprising such nucleic acid constructs or expression vector.

Further aspects and embodiments of the invention are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Sequence alignment of humanization variants of the anti-PSMAVHH JVZ-007. LV1044 is the original llama-derived sequence; the othersequences are humanized variants.

FIG. 2 : Schematic representation of the half-life extended format ofthe bispecific VHHs. Fc silencing mutations are indicated by theasterisks. The sequence of the modified hinge is indicated next to thefigure, as well as the CH3 mutations that confer the KiH mediatedhetero-dimerization. HC: heavy chain.

FIG. 3 : T cell activation assay (degranulation) testing the potency ofhumanization variants in inducing T cell degranulation. Upper panel:Titration of bispecific constructs using PSMA-positive LNCaP cells astarget cells. Lower panel: a single high (10 nM) concentration ofbispecific constructs used in a degranulation assay using PC-3 cells astarget cells. 5C8 indicates the anti-Vγ9Vδ2 TCR-specific VHH and 5C8var1is the humanized variant thereof.

FIG. 4 : Antibody induced-, Vγ9Vδ2 T cell mediated cytotoxicity of LNCaPcells.

FIG. 5 : Binding of LV1050-Fc x 5C8var1-Fc to different cells/cell linesin FACS. Upper panel: Binding to PSMA-positive cell line LNCaP and tothe PSMA-negative line PC-3. Lower panel: binding to Vγ9Vδ2T cells.

FIG. 6 : Target dependent, LV1050-Fc x 5C8var1-Fc-induced Vγ9Vδ2 T cellactivation (upper panel) and cytotoxicity (lower panel).

FIG. 7 : Expression levels of PSMA and CD277 on tumor tissue and normalprostate tissue and frequency (as part of the total CD3+ population) ofVγ9Vδ2-T cells in the tissues.

FIG. 8 : Cytotoxicity (24-hour assay) of prostate tumor cells induced byVγ9Vδ2T cells and LV1044-5C8 (“LAVA compound”). *** p<0.05. The numberof tumor cells (upper panel) or normal cells (lower panel) withouttreatment was put at 100%.

FIG. 9 : Interaction mapped with crosslinks between PSMA andLV1050-5C8var1-no-C-tag. Numbers indicate the amino acid position in thePSMA construct with SEQ ID NO:30, which is based on UniProt ID Q04609,but with a different signal peptide. Position numbers in the PSMAconstruct correspond to the numbering in UniProt ID Q04609 as follows:Position in construct=position in UniProt ID Q04609+41. The PSMAfragments shown are set forth in SEQ ID NOs:33-36.

FIG. 10 : Degranulation assay using expanded Vγ9Vδ2-T cells and PSMApositive tumor-derived cell lines. Degranulation was measured usingprimary, expanded Vγ9Vδ2-T cells co-cultured with differentPSMA-expressing cell lines (LNCaP, 22Rv1 and VCaP cells). The percentageof CD107a positive (degranulating) cells in the CD3-Vγ9 gate is plottedas a function of the LV1050-Fc x 5C8var1-Fc concentration.

FIG. 11 : Cytotoxicity assay using expanded Vγ9Vδ2-T cells and PSMApositive- or PSMA negative cells. Cytotoxicity was measured usingprimary, expanded Vγ9Vδ2-T cells cocultured with (A) LNCaP cells(PSMA-positive cells), versus LNCaP.koPSMA cells (PSMA-negative cells)or (B) different PSMA-expressing cell lines (LNCaP, 22Rv1 and VCaPcells). Cell death of target cells was determined by the activity in themedium of a defined intracellular protease using the CytoTox-Glo™ Assay.

FIG. 12 : Degranulation of Vγ9Vδ2-T cells endogenously present inprostate tumor or normal (non-malignant) tissue. Prostate tumor (A) andnormal (non-malignant) prostate tissue (B) cell suspensions wereincubated for 4 hours with a fluorescently labeled CD107a mAb with orwithout 50 nM of LV1050-Fc x 5C8var1-Fc, and the percentage ofCD45+/CD3+/Vγ9+/Vδ2+/CD107a+ cells (indicated as CD107a expression (% ofEpCAM−/CD45+/CD3+/Vγ9+/Vδ2+)) was determined by flow cytometry. Theinduction of CD107a expression by LV1050-Fc x 5C8var1-Fc was expressedrelative to the background (medium only) expression. **P<0.01, pairedT-test; ns=not significant.

FIG. 13 : Degranulation of Vγ9Vδ2-T cells present in autologous PMBCupon co-culture with patient-derived prostate tumor or normal(non-malignant) prostate tissue. Prostate tumor (A) and normal(non-malignant) prostate tissue (B) cell suspensions were incubated for4 hours with a fluorescently labeled CD107a mAb with or without 50 nM ofLV1050-Fc x 5C8var1-Fc in the presence of autologous PBMC, and thepercentage of CD45+/CD3+/Vγ9+/Vδ2+/CD107a+ cells (indicated as CD107aexpression (% of EpCAM−/CD45+/CD3+/Vγ9+/Vδ2+) was determined by flowcytometry. The induction of CD107a expression by LV1050-Fc x 5C8var1-Fcwas expressed relative to the background (medium only) expression.*P<0.05, paired-T test; ns=not significant.

FIG. 14 : Tumor cell lysis mediated by LV1050-Fc x 5C8var1-Fc in thepresence of autologous PBMC. Single cell suspensions from prostate tumortissue (A) or normal (non-malignant) prostate tissue (B) were culturedwith autologous PBMC in a 10:1 E:T ratio, with or without 50 nM ofLV1050-Fc x 5C8var1-Fc and incubated for 24 hours. Cytotoxicity wasdetermined by flow cytometry using 7-AAD-exclusion, and expressed as thepercentage of lysis relative to ‘target cells plus PBMC’. Shown are meanand S.E.M (n=3), *P<0.05.

FIG. 15 : The combination of LV1050-Fc x 5C8var1-Fc and PBMC inhibitstumor growth in mice inoculated with 22Rv1 prostate carcinoma cells. NCGmice (n=4-6/group) were injected subcutaneously with 22Rv1 prostatecancer cells alone, or in combination with PBMC from two healthy donors.On days 0, 7, 14 and 21, LV1050-Fc x 5C8var1-Fc (0.2 or 2 mg/kg) or PBSwas injected IV. Twice a week, tumor sizes were measured using calipers.Mice were sacrificed when the mean tumor volume of a group exceeded2,000 mm³. Tumor volumes (mm³; mean t SEM) are plotted as a function ofthe days after 22Rv1 prostate cancer cell inoculation. Statisticalanalysis (ANOVA (analysis of variance) with Dunnett's post hoc test)showed that using PBMC from donor #1, LV1050-Fc x 5C8var1-Fcadministered at 0.2 or 2 mg/kg resulted in a statistically significantreduction (***P<0.001) in the tumor growth rate, with TGI values on day34 of 91% and 78% respectively. Using PBMC from donor #2, LV1050-Fc x5C8var1-Fc administered at 2 mg/kg resulted in a significant antitumoreffect (*P<0.01), with a TGI value of 52% on day 41.

FIG. 16 : Pharmacokinetics of LV1050-Fc x 5C8var1-Fc in human FcRntransgenic mice after a single IV dose. LV1050-Fc x 5C8var1-Fc and ahumanized IgG control antibody were co-administered intravenously at 2mg/kg, 5 mg/kg or 10 mg/kg per group in human FcRn transgenic mice (n=4per group). The upper graph shows the concentration of LV1050-Fc x5C8var1-Fc over time for the three groups of mice dosed with differentconcentrations of the antibody. The bottom graph shows LV1050-Fc x5C8var1-Fc terminal half-life calculations obtained in the three dosagegroups (using PK Solutions software to analyze the ELISA data).LV1050-Fc x 5C8var1-Fc half-lives calculated for each groupco-administered with IgG control antibody were comparable andcorresponded to 139.9±10.2 hours (at 2 mg/kg), 160.2±13.4 hours (at 5mg/kg) and 171.7±10.6 hours (at 10 mg/kg).

FIG. 17 : Pharmacokinetics of different doses of LV1050-Fc x 5C8var1-Fcin NHP after a single IV dose. Plasma concentrations of LV1050-Fc x5C8var1-Fc were measured pre-dose and then at 0.5, 1, 2, 4, 8, 24, 72,120, 168, 336, 504 and 528 hours after administration. The plasmaconcentration of LV1050-Fc x 5C8var1 (Y-axis) is given as a function oftime.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “human PSMA”, when used herein, refers to the humanProstate-Specific Membrane Antigen protein, also known as glutamatecarboxypeptidase 2 (EC:3.4.17.21), cell growth-inhibiting gene 27protein, Folate hydrolase 1, Folylpoly-gamma-glutamate carboxypeptidase(FGCP), glutamate carboxypeptidase II (GCPII), membrane glutamatecarboxypeptidase (mGCP), N-acetylated-alpha-linked acidic dipeptidase I(NAALADase I) or pteroylpoly-gamma-glutamate carboxypeptidase(UniProtKB-Q04609 (FOLH1_HUMAN)), Isoform I, set forth in SEQ ID NO:24.

The term “human Vβ2”, when used herein, refers to the TRDV2 protein, Tcell receptor delta variable 2 (UniProtKB-AOJD36 (AOJD36_HUMAN) gives anexample of a Vβ2 sequence).

The term “human Vγ9”, when used herein, refers to the TRGV9 protein, Tcell receptor gamma variable 9 (UniProtKB-Q99603_HUMAN gives an exampleof a Vγ9 sequence).

The term “antibody” is intended to refer to an immunoglobulin molecule,a fragment of an immunoglobulin molecule, or a derivative of eitherthereof, which has the ability to specifically bind to an antigen undertypical physiological conditions with a half-life of significant periodsof time, such as at least about 30 minutes, at least about 45 minutes,at least about one hour, at least about two hours, at least about fourhours, at least about 8 hours, at least about 12 hours, about 24 hoursor more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc.,or any other relevant functionally-defined period (such as a timesufficient to induce, promote, enhance, and/or modulate a physiologicalresponse associated with antibody binding to the antigen and/or timesufficient for the antibody to recruit an effector activity). Theantigen-binding region (or antigen-binding domain) which interacts withan antigen may comprise variable regions of both the heavy and lightchains of the immunoglobulin molecule or may be a single-domainantigen-binding region, e.g. a heavy chain variable region only. Theconstant regions of an antibody, if present, may mediate the binding ofthe immunoglobulin to host tissues or factors, including various cellsof the immune system (such as effector cells and T cells) and componentsof the complement system such as C1q, the first component in theclassical pathway of complement activation. In some embodiments,however, the Fc region of the antibody has been modified to becomeinert, “inert” means an Fc region which is at least not able to bind anyFcγ Receptors, induce Fc-mediated cross-linking of FcRs, or induceFcR-mediated cross-linking of target antigens via two Fc regions ofindividual antibodies. In a further embodiment, the inert Fc region isin addition not able to bind C1q. In one embodiment, the antibodycontains mutations at positions 234 and 235 (Canfield and Morrison(1991) J Exp Med 173:1483), e.g. a Leu to Phe mutation at position 234and a Leu to Glu mutation at position 235. In another embodiment, theantibody contains a Leu to Ala mutation at position 234, a Leu to Alamutation at position 235 and a Pro to Gly mutation at position 329. Inanother embodiment, the antibody contains a Leu to Phe mutation atposition 234, a Leu to Glu mutation at position 235 and an Asp to Ala atposition 265.

The Fc region of an immunoglobulin is defined as the fragment of anantibody which would be typically generated after digestion of anantibody with papain which includes the two CH2-CH3 regions of animmunoglobulin and a connecting region, e.g. a hinge region. Theconstant domain of an antibody heavy chain defines the antibody isotype,e.g. IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc-regionmediates the effector functions of antibodies with cell surfacereceptors called Fc receptors and proteins of the complement system.

The term “hinge region” as used herein is intended to refer to the hingeregion of an immunoglobulin heavy chain. Thus, for example the hingeregion of a human IgG1 antibody corresponds to amino acids 216-230according to the EU numbering.

The term “CH2 region” or “CH2 domain” as used herein is intended torefer to the CH2 region of an immunoglobulin heavy chain. Thus, forexample the CH2 region of a human IgG1 antibody corresponds to aminoacids 231-340 according to the EU numbering. However, the CH2 region mayalso be any of the other subtypes as described herein.

The term “CH3 region” or “CH3 domain” as used herein is intended torefer to the CH3 region of an immunoglobulin heavy chain. Thus, forexample the CH3 region of a human IgG1 antibody corresponds to aminoacids 341-447 according to the EU numbering. However, the CH3 region mayalso be any of the other subtypes as described herein.

Reference to amino acid positions in the Fc region/Fc domain in thepresent invention is according to the EU-numbering (Edelman et al., ProcNatl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences ofproteins of immunological interest. 5th Edition-1991 NIH Publication No.91-3242).

As indicated above, the term antibody as used herein, unless otherwisestated or clearly contradicted by context, includes fragments of anantibody that retain the ability to specifically bind to the antigen. Ithas been shown that the antigen-binding function of an antibody may beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antibody” include (i) a Fab′ orFab fragment, i.e. a monovalent fragment consisting of the VL, VH, CLand CH1 domains, or a monovalent antibody as described in WO2007059782;(ii) F(ab′)2 fragments, i.e. bivalent fragments comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting essentially of the VH and CH1 domains; and (iv) a Fvfragment consisting essentially of the VL and VH domains of a single armof an antibody. Furthermore, although the two domains of the Fvfragment, VL and VH, are coded for by separate genes, they may bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain antibodies orsingle chain Fv (scFv), see for instance Bird et al., Science 242,423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Suchsingle chain antibodies are encompassed within the term antibody unlessotherwise indicated by context. Although such fragments are generallyincluded within the meaning of antibody, they collectively and eachindependently are unique features of the present invention, exhibitingdifferent biological properties and utility. The term antibody, unlessspecified otherwise, also includes polyclonal antibodies, monoclonalantibodies (mAbs), chimeric antibodies and humanized antibodies, andantibody fragments provided by any known technique, such as enzymaticcleavage, peptide synthesis, and recombinant techniques.

In some embodiments of the antibodies of the invention, the firstantigen-binding region or the antigen-binding region, or both, is asingle domain antibody. Single domain antibodies (sdAb, also calledNanobody®, or VHH) are well known to the skilled person, see e.g.Hamers-Casterman et al. (1993) Nature 363:446, Roovers et al. (2007)Curr Opin Mol Ther 9:327 and Krah et al. (2016) ImmunopharmacolImmunotoxicol 38:21. Single domain antibodies comprise a single CDR1, asingle CDR2 and a single CDR3. Examples of single domain antibodies arevariable fragments of heavy-chain-only antibodies, antibodies thatnaturally do not comprise light chains, single domain antibodies derivedfrom conventional antibodies, and engineered antibodies. Single domainantibodies may be derived from any species including mouse, human,camel, llama, shark, goat, rabbit, and cow. For example, naturallyoccurring VHH molecules can be derived from antibodies raised inCamelidae species, for example in camel, dromedary, llama, alpaca andguanaco. Like a whole antibody, a single domain antibody is able to bindselectively to a specific antigen. Single domain antibodies may containonly the variable domain of an immunoglobulin chain, i.e. CDR1, CDR2 andCDR3 and framework regions.

The term “immunoglobulin” as used herein is intended to refer to a classof structurally related glycoproteins consisting of two pairs ofpolypeptide chains, one pair of light (L) chains and one pair of heavy(H) chains, all four potentially inter-connected by disulfide bonds. Theterm “immunoglobulin heavy chain”, “heavy chain of an immunoglobulin” or“heavy chain” as used herein is intended to refer to one of the chainsof an immunoglobulin. A heavy chain is typically comprised of a heavychain variable region (abbreviated herein as VH) and a heavy chainconstant region (abbreviated herein as CH) which defines the isotype ofthe immunoglobulin. The heavy chain constant region typically iscomprised of three domains, CH1, CH2, and CH3. The heavy chain constantregion further comprises a hinge region. Within the structure of theimmunoglobulin (e.g. IgG), the two heavy chains are inter-connected viadisulfide bonds in the hinge region. Equally to the heavy chains, eachlight chain is typically comprised of several regions; a light chainvariable region (VL) and a light chain constant region (CL).Furthermore, the VH and VL regions may be subdivided into regions ofhypervariability (or hypervariable regions which may be hypervariable insequence and/or form structurally defined loops), also termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FRs). Each VH and VLis typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. CDR sequences may be determined by use ofvarious methods, e.g. the methods provided by Choitia and Lesk (1987) J.Mol. Biol. 196:901 or Kabat et al. (1991) Sequence of protein ofimmunological interest, fifth edition. NIH publication. Various methodsfor CDR determination and amino acid numbering can be compared onwww.abysis.org (UCL).

The term “isotype” as used herein, refers to the immunoglobulin(sub)class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM)or any allotype thereof, such as IgG1m(za) and IgG1m(f) that is encodedby heavy chain constant region genes. Each heavy chain isotype can becombined with either a kappa (κ) or lambda (A) light chain. An antibodyof the invention can possess any isotype.

The term “parent antibody”, is to be understood as an antibody which isidentical to an antibody according to the invention, but wherein theparent antibody does not have one or more of the specified mutations. A“variant” or “antibody variant” or a “variant of a parent antibody” ofthe present invention is an antibody molecule which comprises one ormore mutations as compared to a “parent antibody”. Amino acidsubstitutions may exchange a native amino acid for another naturallyoccurring amino acid, or for a non-naturally occurring amino acidderivative. The amino acid substitution may be conservative ornon-conservative. In the context of the present invention, conservativesubstitutions may be defined by substitutions within the classes ofamino acids reflected in one or more of the following three tables:

Amino Acid Residue Classes for Conservative Substitutions

Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), andHis (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), andGln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L),and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

Alternative Conservative Amino Acid Residue Substitution Classes

1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

Alternative Physical and Functional Classifications of Amino AcidResidues

Alcohol group-containing residues S and T Aliphatic residues I, L, V,and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turn A,C, D, E, G, H, K, N, Q, R, S, formation P, and T Flexible residues Q, T,K, S, G, N, D, E, and RIn the context of the present invention, a substitution in a variant isindicated as:

Original amino acid-position-substituted amino acid;

The three-letter code, or one letter code, are used, including the codesXaa and X to indicate amino acid residue. Accordingly, the notation“T366W” means that the variant comprises a substitution of threoninewith tryptophan in the variant amino acid position corresponding to theamino acid in position 366 in the parent antibody.

Furthermore, the term “a substitution” embraces a substitution into anyone of the other nineteen natural amino acids, or into other aminoacids, such as non-natural amino acids. For example, a substitution ofamino acid T in position 366 includes each of the followingsubstitutions: 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L,366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y.

The term “full-length antibody” when used herein, refers to an antibodywhich contains all heavy and light chain constant and variable domainscorresponding to those that are normally found in a wild-type antibodyof that isotype.

The term “chimeric antibody” refers to an antibody wherein the variableregion is derived from a non-human species (e.g. derived from rodents)and the constant region is derived from a different species, such ashuman. Chimeric antibodies may be generated by genetic engineering.Chimeric monoclonal antibodies for therapeutic applications aredeveloped to reduce antibody immunogenicity.

The term “humanized antibody” refers to a genetically engineerednon-human antibody, which contains human antibody constant domains andnon-human variable domains modified to contain a high level of sequencehomology to human variable domains. This can be achieved by grafting ofthe six non-human antibody complementarity-determining regions (CDRs),which together form the antigen binding site, onto a homologous humanacceptor framework region (FR). In order to fully reconstitute thebinding affinity and specificity of the parental antibody, thesubstitution of framework residues from the parental antibody (i.e. thenon-human antibody) into the human framework regions (back-mutations)may be required. Structural homology modeling may help to identify theamino acid residues in the framework regions that are important for thebinding properties of the antibody. Thus, a humanized antibody maycomprise non-human CDR sequences, primarily human framework regionsoptionally comprising one or more amino acid back-mutations to thenon-human amino acid sequence, and, optionally, fully human constantregions. Optionally, additional amino acid modifications, which are notnecessarily back-mutations, may be introduced to obtain a humanizedantibody with preferred characteristics, such as affinity andbiochemical properties. Humanization of non-human therapeutic antibodiesis performed to minimize its immunogenicity in man while such humanizedantibodies at the same time maintain the specificity and bindingaffinity of the antibody of non-human origin.

The term “multispecific antibody” refers to an antibody havingspecificities for at least two different, such as at least three,typically non-overlapping, epitopes. Such epitopes may be on the same oron different target antigens. If the epitopes are on different targets,such targets may be on the same cell or different cells or cell types.

The term “bispecific antibody” refers to an antibody havingspecificities for two different, typically non-overlapping, epitopes.Such epitopes may be on the same or different targets. If the epitopesare on different targets, such targets may be on the same cell ordifferent cells or cell types.

Examples of different classes of bispecific antibodies include but arenot limited to (i) IgG-like molecules with complementary CH3 domains toforce heterodimerization; (ii) recombinant IgG-like dual targetingmolecules, wherein the two sides of the molecule each contain the Fabfragment or part of the Fab fragment of at least two differentantibodies; (iii) IgG fusion molecules, wherein full length IgGantibodies are fused to extra Fab fragment or parts of Fab fragment;(iv) Fc fusion molecules, wherein single chain Fv molecules orstabilized diabodies are fused to heavy-chain constant-domains,Fc-regions or parts thereof; (v) Fab fusion molecules, wherein differentFab-fragments are fused together, fused to heavy-chain constant-domains,Fc-regions or parts thereof; and (vi) ScFv- and diabody-based and heavychain antibodies (e.g., domain antibodies, Nanobodies®) whereindifferent single chain Fv molecules or different diabodies or differentheavy-chain antibodies (e.g. domain antibodies, Nanobodies®) are fusedto each other or to another protein or carrier molecule fused toheavy-chain constant-domains, Fc-regions or parts thereof.

Examples of IgG-like molecules with complementary CH3 domains moleculesinclude but are not limited to the Triomab® (Trion Pharma/FreseniusBiotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and theelectrostatically-matched (Amgen, Chugai, Oncomed), the LUZ-Y(Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi:10.1074/jbc.M112.397869. Epub 2012 Nov. 1), DIG-body and PIG-body(Pharmabcine, WO2010134666, WO2014081202), the Strand ExchangeEngineered Domain body (SEEDbody)(EMD Serono), the Biclonics (Merus,WO2013157953), FcΔAdp (Regeneron), bispecific IgG1 and IgG2(Pfizer/Rinat), Azymetric scaffold (Zymeworks/Merck), mAb-Fv (Xencor),bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody®molecules (Genmab).

Examples of recombinant IgG-like dual targeting molecules include butare not limited to Dual Targeting (DT)-Ig (GSK/Domantis, WO2009058383),Two-in-one Antibody (Genentech, Bostrom, et al 2009. Science 323,1610-1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star),Zybodies™ (Zyngenia, LaFleur et al. MAbs. 2013 March-April;5(2):208-18), approaches with common light chain, KABodies (NovImmune,WO2012023053) and CovX-body® (CovX/Pfizer, Doppalapudi, V. R., et al2007. Bioorg. Med. Chem. Lett. 17,501-506).

Examples of IgG fusion molecules include but are not limited to DualVariable Domain (DVD)-Ig (Abbott), Dual domain double head antibodies(Unilever; Sanofi Aventis), IgG-like Bispecific (ImClone/Eli Lilly,Lewis et al. Nat Biotechnol. 2014 February; 32(2):191-8), Ts2Ab(MedImmune/AZ, Dimasi et al. J Mol Biol. 2009 Oct. 30; 393(3):672-92)and BsAb (Zymogenetics, WO2010111625), HERCULES (Biogen Idec), scFvfusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc) and TvAb(Roche).

Examples of Fc fusion molecules include but are not limited to ScFv/FcFusions (Academic Institution, Pearce et al Biochem Mol Biol Int. 1997September; 42(6):1179), SCORPION (Emergent BioSolutions/Trubion,Blankenship J W, et al. AACR 100th Annual meeting 2009 (Abstract #5465);Zymogenetics/BMS, WO2010111625), Dual Affinity Retargeting Technology(Fc-DART™) (MacroGenics) and Dual(ScFv)2-Fab (National Research Centerfor Antibody Medicine—China).

Examples of Fab fusion bispecific antibodies include but are not limitedto F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech),Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) andFab-Fv (UCB-Celltech).

Examples of ScFv-, diabody-based and domain antibodies include but arenot limited to Bispecific T Cell Engager (BiTE®) (Micromet, TandemDiabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™)(MacroGenics), Single-chain Diabody (Academic, Lawrence FEBS Lett. 1998Apr. 3; 425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), HumanSerum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODYmolecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 August;88(6):667-75), dual targeting Nanobodies® (Ablynx, Hmila et al., FASEBJ. 2010), dual targeting heavy chain only domain antibodies.

In the context of antibody binding to an antigen, the terms “binds” or“specifically binds” refer to the binding of an antibody to apredetermined antigen or target (e.g. human PSMA or Vβ2) to whichbinding typically is with an affinity corresponding to a K_(D) of about10⁻⁶ M or less, e.g. 10⁻⁷ M or less, such as about 10⁻⁸ M or less, suchas about 10⁻⁹ M or less, about 10⁻¹⁰ M or less, or about 10⁻¹¹ M or evenless, e.g. when determined using flow cytometry as described in theExamples herein. Alternatively, apparent K_(D) values can be determinedusing for instance surface plasmon resonance (SPR) technology in aBIAcore T200 instrument using the antigen as the ligand and the bindingmoiety or binding molecule as the analyte. Specific binding means thatthe antibody binds to the predetermined antigen with an affinitycorresponding to a K_(D) that is at least ten-fold lower, such as atleast 100-fold lower, for instance at least 1,000-fold lower, such as atleast 10,000-fold lower, for instance at least 100,000-fold lower thanits affinity for binding to a non-specific antigen (e.g., BSA, casein)other than the predetermined antigen or a closely-related antigen. Thedegree with which the affinity is lower is dependent on the K_(D) of thebinding moiety or binding molecule, so that when the K_(D) of thebinding moiety or binding molecule is very low (that is, the bindingmoiety or binding molecule is highly specific), then the degree withwhich the affinity for the antigen is lower than the affinity for anon-specific antigen may be at least 10,000-fold. The term “K_(D)” (M),as used herein, refers to the dissociation equilibrium constant of aparticular interaction between the antigen and the binding moiety orbinding molecule.

In the context of the present invention, “competition” or “able tocompete” or “competes” refers to any detectably significant reduction inthe propensity for a particular binding molecule (e.g. a PSMA antibody)to bind a particular binding partner (e.g. PSMA) in the presence ofanother molecule (e.g. a different PSMA antibody) that binds the bindingpartner. Typically, competition means an at least about 25 percentreduction, such as an at least about 50 percent, e.g. an at least about75 percent, such as an at least 90 percent reduction in binding, causedby the presence of another molecule, such as an antibody, as determinedby, e.g., ELISA analysis or flow cytometry using sufficient amounts ofthe two or more competing molecules, e.g. antibodies. Additional methodsfor determining binding specificity by competitive inhibition may befound in for instance Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988),Colligan et al., eds., Current Protocols in Immunology, GreenePublishing Assoc, and Wiley InterScience N. Y., (1992, 1993), andMuller, Meth. Enzymol. 92, 589-601 (1983)). In one embodiment, theantibody of the present invention binds to the same epitope on PSMA asantibody LV1050 and/or to the same epitope on Vδ2 as antibody 5C8 or6H4. Methods for determining the epitope of a binding molecule, such asan antibody, are known in the art.

The terms “first” and “second” antigen-binding regions when used hereindo not refer to their orientation/position in the antibody, i.e. theyhave no meaning with regard to the N- or C-terminus. The terms “first”and “second” only serve to correctly and consistently refer to the twodifferent antigen-binding regions in the claims and the description.

“Capable of binding a Vγ9Vδ2-TCR” means that the binding molecule canbind a Vγ9Vδ2-TCR, but does not exclude that the binding molecule bindsto one of the separate subunits in the absence of the other subunit,i.e. to the Vγ9 chain alone or to the Vδ2 chain alone. For example,antibody 5C8 is an antibody that binds the Vγ9Vδ2-TCR, but also bindsthe Vδ2 chain when the Vδ2 chain is expressed alone.

The term does also not exclude that the antibody is specific for thecombination of the two chains.

“% sequence identity”, when used herein, refers to the number ofidentical nucleotide or amino acid positions shared by differentsequences (i.e., % identity=# of identical positions/total # ofpositions×100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment. Thepercent identity between two nucleotide or amino acid sequences may e.g.be determined using the algorithm of E. Meyers and W. Miller, Comput.Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

FURTHER ASPECTS AND EMBODIMENTS OF THE INVENTION

As described above, in a first main aspect, the invention relates to amultispecific antibody comprising a first antigen-binding region capableof binding human PSMA and a second antigen-binding region capable ofbinding a human Vγ9Vβ2 T cell receptor.

In one embodiment, the multispecific antibody is a bispecific antibody.In another embodiment, the first antigen-binding region is asingle-domain antibody. In another embodiment, the secondantigen-binding region is a single-domain antibody. In a furtherembodiment, both the first antigen-antigen binding region and the secondantigen-binding region are single-domain antibodies. In a furtherembodiment, the multispecific antibody is a bispecific antibody, whereinthe first antigen-binding region is a single-domain antibody and thesecond antigen-binding region is a single-domain antibody,

In one embodiment, the multispecific antibody competes (i.e. is able tocompete) for binding to human PSMA with an antibody having the sequenceset forth in SEQ ID NO:2, preferably the multispecific antibody bindsthe same epitope on human PSMA as an antibody having the sequence setforth in SEQ ID NO:2. In one embodiment, the multispecific antibodybinds to an epitope on human PSMA which comprises one or more, or all,of the following amino acid residues: R190, S197, R204, S317, R320,S322, K324, H618, S631, K729, R730 and Y733, wherein numbering isaccording to UniProt sequence Q04609-1. In a further embodiment, thefirst antigen-binding region comprises the VH CDR1 sequence set forth inSEQ ID NO:14, the VH CDR2 sequence set forth in SEQ ID NO: 15 and the VHCDR3 sequence set forth in SEQ ID NO:16.

In a further embodiment, the first antigen-binding region is humanized,wherein preferably the first antigen-binding region comprises orconsists of:

-   -   the sequence set forth in SEQ ID NO:2, or    -   a sequence having at least 90%, such as least 92%, e.g. at least        94%, such as at least 96%, e.g. at least 98% sequence identity        to the sequence set forth in SEQ ID NO:2.

As described above, the multispecific antibody of the inventioncomprises a second antigen-binding region capable of binding a humanVγ9Vβ2-T cell receptor. In one embodiment, the multispecific antibody isable to activate human Vγ9Vβ2 T cells. The activation of the Vγ9Vδ2 Tcells may be measured through gene-expression and/or (surface) markerexpression (e.g., activation markers, such as CD25, CD69, or CD107a)and/or secretory protein (e.g., cytokines or chemokines) profiles. In apreferred embodiment, the multispecific antibody is able to induceactivation (e.g. upregulation of CD69 and/or CD25 expression) resultingin degranulation marked by an increase in CD107a expression (see e.g.Example 5) and/or cytokine production (e.g. TNFα, IFNγ) by Vγ9Vδ2 Tcells. Preferably, a multispecific antibody of the present invention isable to increase the number of cells positive for CD107a at least2-fold, such as at least 5-fold, when tested as described in Example 5herein. In another preferred embodiment, the multispecific antibody ofthe invention has an EC50 value for increasing the percentage of CD107apositive cells of 50 pM or less, such as 25 pM or less, e.g. 20 pM orless, such as 15 pM or less, e.g. 10 pM or less, or even 5 pM or less,such as 2 pM or less or 1 pM or less when tested using Vγ9Vδ2 T cellsand LNCaP target cells as described herein in Example 5.

In one embodiment of the multispecific antibody of the invention, themultispecific antibody is capable of binding to human Vβ2. Vδ2 is partof the delta chain of the Vγ9Vδ2-TCR. An antibody capable of binding tohuman Vβ2 may bind an epitope that is entirely located within the Vβ2region or bind an epitope that is a combination of residues in Vβ2region and the constant region of the delta chain. In anotherembodiment, the multispecific antibody is capable of binding to humanVγ9. Vγ9 is part of the gamma chain of Vγ9Vδ2-TCR. An antibody capableof binding to human Vγ9 may bind an epitope that is entirely locatedwithin the Vγ9 region or bind an epitope that is a combination ofresidues in Vγ9 region and the constant region of the gamma chain.Several such antibodies which bind to Vδ2 or Vγ9 have been described inWO2015156673 and their antigen-binding regions at least the CDRsequences thereof can be incorporated in the multispecific antibody ofthe invention. Other examples of antibodies from which aVγ9Vδ2-TCR-binding region might be derived are TCR Vγ9 antibody 7A5(ThermoFisher) (Oberg et al. (2014) Cancer Res 74:1349) and antibodiesB1.1 (ThermoFisher) and 5A6.E9 (ATCC HB 9772), both described in Neumanet al. (2016) J Med Prim 45:139.

In one embodiment, the multispecific antibody competes for binding tohuman Vβ2 with an antibody having the sequence set forth in SEQ ID NO:5or competes for binding to human Vβ2 with an antibody having thesequence set forth in SEQ ID NO:20. In a further embodiment, themultispecific antibody binds the same epitope on human Vβ2 as anantibody having the sequence set forth in SEQ ID NO:5 or binds the sameepitope on human Vβ2 as an antibody having the sequence set forth in SEQID NO:20.

In one embodiment of the multispecific antibody of the invention, thesecond antigen-binding region comprises the VH CDR1 sequence set forthin SEQ ID NO: 17, the VH CDR2 sequence set forth in SEQ ID NO:18 and theVH CDR3 sequence set forth in SEQ ID NO: 19 or comprises the VH CDR1sequence set forth in SEQ ID NO:21, the VH CDR2 sequence set forth inSEQ ID NO:22 and the VH CDR3 sequence set forth in SEQ ID NO:23.

In one embodiment of the multispecific antibody of the invention, thesecond antigen-binding region is humanized.

In a further embodiment, the second antigen-binding region comprises orconsists of

-   -   the sequence set forth in SEQ ID NO:5, or    -   a sequence having at least 90%, such as least 92%, e.g. at least        94%, such as at least 96%, e.g. at least 98% sequence identity        to the sequence set forth in SEQ ID NO:5.

In one embodiment of the multispecific antibody of the invention, thefirst antigen-binding region comprises the VH CDR1 sequence set forth inSEQ ID NO: 14, the VH CDR2 sequence set forth in SEQ ID NO:15 and the VHCDR3 sequence set forth in SEQ ID NO: 16 and wherein the secondantigen-binding region comprises the VH CDR1 sequence set forth in SEQID NO:17, the VH CDR2 sequence set forth in SEQ ID NO: 18 and the VHCDR3 sequence set forth in SEQ ID NO: 19.

In another embodiment of the multispecific antibody of the invention,the first antigen-binding region comprises the VH CDR1 sequence setforth in SEQ ID NO: 14, the VH CDR2 sequence set forth in SEQ ID NO:15and the VH CDR3 sequence set forth in SEQ ID NO: 16 and wherein thesecond antigen-binding region comprises the VH CDR1 sequence set forthin SEQ ID NO:21, the VH CDR2 sequence set forth in SEQ ID NO:22 and theVH CDR3 sequence set forth in SEQ ID NO:23.

In one embodiment, the multispecific antibody of the invention iscapable of mediating killing of PSMA-expressing cells, e.g. LNCaP cells,22Rv1 cells or VCaP cells through activation of Vγ9Vδ2 T cells.Preferably, the antibody is capable of inducing killing of LNCaP cellsthrough activation of Vγ9Vδ2 T cells with an EC50 value of 50 pM orless, such as 25 pM or less, e.g. 20 pM or less, such as 15 pM or less,e.g. 10 pM or less, or even 5 pM or less, such as 2 pM or less or 1 pMor less when tested as described in Example 6 herein.

In another embodiment, the antibody is capable of inducing killing ofLNCaP, 22Rv1 or VCaP cells through activation of Vγ9Vδ2 T cells with anEC50 value of 50 pM or less, such as 25 pM or less, e.g. 20 pM or less,such as 15 pM or less when tested after 24 hours as described in Example13 herein, preferably both at a 1:1 and a 1:10 effector to target cellratio.

In another embodiment, the multispecific antibody of the invention iscapable of binding to the PSMA positive prostate cancer cell line LNCaPwith an EC50 of 50 nM or less, such as 20 nM or less, e.g. 10 nM ofless, when tested as described in Example 7 herein. In anotherembodiment, the multispecific antibody of the invention is capable ofbinding to Vγ9Vδ2 T cells with an EC50 of 10 nM or less, such as 5 nM orless, e.g. 2 nM of less, when tested as described in Example 7 herein.In another embodiment, the multispecific antibody of the invention iscapable of binding to recombinant human PSMA protein with a KD value of100 nM or less, such as 50 nM or less, when tested as described inExample 11 herein. In another embodiment, the multispecific antibody ofthe invention is capable of binding to human Vγ9Vδ2-Fc with a KD valueof 10 nM or less, such as 5 nM or less, e.g. 2 nM or less, such as 1 nMor less when tested as described in Example 11 herein.

In a further embodiment, the multispecific antibody is capable ofmediating killing of human PSMA-expressing cells from a prostate cancerpatient. Killing of human PSMA-expressing cells from a prostate cancerpatient may e.g. be determined as described in Example 10 herein. In oneembodiment, the multispecific antibody of the invention is capable ofmediating specific cell death of more than 25%, such as more than 50%,at a concentration of 50 nM, as determined in the assays described inExample 10 or Example 14 herein.

In a further embodiment, the multispecific antibody is not capable ofmediating killing of PSMA-negative cells, such as PSMA negative humancells. In another embodiment, the multispecific antibody does not induceIL-2, IL-4, IL-6, IL-10 or TNF a in whole blood from healthy donors atconcentrations up to 280 nM, when tested as described in Example 16herein. In another embodiment, the multispecific antibody induces morethan 10-fold less IL-8 and/or more than 50-fold less IFNγ than Campath®in whole blood from healthy donors when tested as described in Example16 herein.

In one embodiment, the first antigen-binding region and the secondantigen-binding region are covalently linked to each other via a peptidelinker, e.g. a linker having a length of from 1 to 20 amino acids, e.g.from 1 to 10 amino acids, such as 2, 3, 4, 5, 6, 7, 8 or 10 amino acids.In one embodiment, the peptide linker comprises or consists of thesequence GGGGS, set forth in SEQ ID NO:6.

In some embodiments, the first antigen-binding region capable of bindinghuman PSMA is located N-terminally of the second antigen-binding regioncapable of binding a human Vγ9Vβ2 T cell receptor.

In one embodiment of the invention, the multispecific antibody furthercomprises a half-life extension domain. In one embodiment, themultispecific antibody has a terminal half-life that is longer thanabout 168 hours when administered to a human subject. Most preferablythe terminal half-life is 336 hours or longer. The “terminal half-life”of an antibody, when used herein refers to the time taken for the serumconcentration of the polypeptide to be reduced by 50%, in vivo in thefinal phase of elimination.

In one embodiment, the multispecific antibody comprises an Fc region.Various method for making bispecific antibodies have been described inthe art, e.g. reviewed by Brinkmann and Kontermann (2017) MAbs 9:182. Inone embodiment of the present invention, the Fc region is a heterodimercomprising two Fc polypeptides, wherein the first antigen-binding regionis fused to the first Fc polypeptide and the second antigen-bindingregion is fused to the second Fc polypeptide and wherein the first andsecond Fc polypeptides comprise asymmetric amino acid mutations thatfavor the formation of heterodimers over the formation of homodimers(see e.g. Ridgway et al. (1996) ‘Knobs-into-holes’ engineering ofantibody CH3 domains for heavy chain heterodimerization. Protein Eng9:617). In a further embodiment hereof, the CH3 regions of the Fcpolypeptides comprise said asymmetric amino acid mutations, preferablythe first Fc polypeptide comprises a T366W substitution and the secondFc polypeptide comprises T366S, L368A and Y407V substitutions, or viceversa, wherein the amino acid positions correspond to human IgG1according to the EU numbering system. In a further embodiment, thecysteine residues at position 220 in the first and second Fcpolypeptides have been deleted or substituted, wherein the amino acidposition corresponds to human IgG1 according to the EU numbering system.In a further embodiment, the region comprises the sequence set forth inSEQ ID NO:10.

In some embodiments, the first and/or second Fc polypeptides containmutations that render the antibody inert, i.e. unable to mediateeffector functions. In one embodiment, the first and second Fcpolypeptides comprise a mutation at position 234 and/or 235, preferablythe first and second Fc polypeptide comprise an L234F and an L235Esubstitution, wherein the amino acid positions correspond to human IgG1according to the EU numbering system.

In a preferred embodiment, the first antigen-binding region comprisesthe sequence set forth in SEQ ID NO:2, the second antigen-binding regioncomprises the sequence set forth in SEQ ID NO:5 and

-   -   the first Fc polypeptide comprises the sequence set forth in SEQ        ID NO:12 and the second Fc polypeptide comprises the sequence        set forth in SEQ ID NO:13, or    -   the first Fc polypeptide comprises the sequence set forth in SEQ        ID NO:12 and the second Fc polypeptide comprises the sequence        set forth in SEQ ID NO: 13.

In a further embodiment, the antibody comprises or consists of thesequences set forth in SEQ ID NO:25 and SEQ ID NO:26.

In a further main aspect, the invention relates to a pharmaceuticalcomposition comprising a multispecific antibody according to theinvention as described herein and a pharmaceutically-acceptableexcipient.

Antibodies may be formulated with pharmaceutically-acceptable excipientsin accordance with conventional techniques such as those disclosed in(Rowe et al., Handbook of Pharmaceutical Excipients, 2012 June, ISBN9780857110275). The pharmaceutically-acceptable excipient as well as anyother carriers, diluents or adjuvants should be suitable for theantibodies and the chosen mode of administration. Suitability forexcipients and other components of pharmaceutical compositions isdetermined based on the lack of significant negative impact on thedesired biological properties of the chosen antibody or pharmaceuticalcomposition of the present invention (e.g., less than a substantialimpact (10% or less relative inhibition, 5% or less relative inhibition,etc.) upon antigen binding).

A pharmaceutical composition may include diluents, fillers, salts,buffers, detergents (e.g., a nonionic detergent, such as Tween-20 orTween-80), stabilizers (e.g., sugars or protein-free amino acids),preservatives, tissue fixatives, solubilizers, and/or other materialssuitable for inclusion in a pharmaceutical composition. Furtherpharmaceutically-acceptable excipients include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption-delaying agentsand the like that are physiologically compatible with an antibody of thepresent invention.

In one embodiment, the pharmaceutical composition comprises amultispecific antibody of the invention (preferably an antibodycomprising an Fc region), a buffer and an antioxidant, wherein the pH ofthe composition is between 5.4 and 7.4, such as between 5.4 and 6.1.

In a further embodiment, the pharmaceutical composition comprises amultispecific antibody of the invention, a buffer and methionine,wherein the pH of the composition is between 5.4 and 7.4, such asbetween 5.4 and 6.1.

In a further embodiment, the pharmaceutical composition comprises amultispecific antibody of the invention, a buffer, sucrose, polysorbate80 and methionine, wherein the pH of the composition is between 5.4 and7.4, such as between 5.4 and 6.1.

In a further embodiment, the pharmaceutical composition comprises amultispecific antibody of the invention, a histidine or sodium acetatebuffer, sucrose, polysorbate 80 and methionine, wherein the pH of thecomposition is between 5.4 and 7.4, such as between 5.4 and 6.1.

Preferably, the buffer concentration is between 5 and 25 mM, such as 10mM. Preferably, the sucrose concentration is between 100 and 500 mM,such as between 250 mM and 300 mM, e.g. 280 mM. Preferably, thepolysorbate 80 concentration is between 0.005% and 0.1%, such between0.01% and 0.05%, e.g. 0.02%. Preferably, the methionine concentration isbetween 0.2 mM and 5 mM, such between 0.5 mM and 2 mM, e.g. 1 mM.Preferably, the multispecific antibody in the composition comprises anFc region.

Thus, in a preferred embodiment, the pharmaceutical compositioncomprises a multispecific antibody of the invention comprising an Fcregion, a histidine or sodium acetate buffer, sucrose, polysorbate 80and methionine, wherein the pH of the composition is between 5.4 and7.4, such as between 5.4 and 6.1.

Thus, in a further preferred embodiment, the pharmaceutical compositioncomprises a multispecific antibody of the invention comprising an Fcregion, a 10 M histidine or sodium acetate buffer, 280 mM sucrose, 0.02%polysorbate 80 and 1 mM methionine, wherein the pH of the composition is5.5 or 6.0.

Preferably, such as Fc region is a heterodimer comprising two Fcpolypeptides, wherein the first antigen-binding region is fused to thefirst Fc polypeptide and the second antigen-binding region is fused tothe second Fc polypeptide and wherein the first and second Fcpolypeptides comprise asymmetric amino acid mutations that favor theformation of heterodimers over the formation of homodimers. Preferably,the CH3 regions of the Fc polypeptides comprise said asymmetric aminoacid mutations, wherein preferably the first Fc polypeptide comprises aT366W substitution and the second Fc polypeptide comprises T366S, L368Aand Y407V substitutions, or vice versa. Furthermore, preferably, thecysteine residues at position 220 in the first and second Fcpolypeptides have been deleted or substituted and/or the first andsecond Fc polypeptides further comprise a mutation at position 234and/or 235, wherein preferably the first and second Fc polypeptidecomprise an L234F and an L235E substitution.

Furthermore, preferably, the multispecific antibody in the compositioncomprises a first antigen-binding region comprising the sequence setforth in SEQ ID NO:2, a second antigen-binding region comprising thesequence set forth in SEQ ID NO:5 and

-   -   the first Fc polypeptide comprises the sequence set forth in SEQ        ID NO: 12 and the second Fc polypeptide comprises the sequence        set forth in SEQ ID NO: 13, or    -   the first Fc polypeptide comprises the sequence set forth in SEQ        ID NO: 12 and the second Fc polypeptide comprises the sequence        set forth in SEQ ID NO: 13 and the pharmaceutical composition        comprises a 10 M histidine or sodium acetate buffer, 280 mM        sucrose, 0.02% polysorbate 80 and 1 mM methionine, wherein the        pH of the composition is 5.5 or 6.0. Preferably, the antibody        concentration is between 0.1 mg/ml and 20 mg/ml, such as between        0.1 and 10 mg/ml, for example between 0.5 mg/ml and 2 mg/ml,        such as 1 mg/ml.

In a further main aspect, the invention relates to the multispecificantibody according to the invention as described herein for use as amedicament.

A multispecific antibody according to the invention enables creating amicroenvironment that is beneficial for killing of tumor cells, inparticular PSMA-positive tumor cells, by Vγ9Vδ2 T cells.

Accordingly, in a preferred embodiment, the multispecific antibody isfor use in the treatment of cancer. In a further preferred embodiment,the multispecific antibody is for use in the treatment of prostatecancer, such as metastatic or non-metastatic prostate cancer. In anotherembodiment, the multispecific antibody is for use in the treatment ofcancers in which PSMA is expressed on the tumor neo-vasculature ortumor-associated endothelial cells of primary or metastatic tumorsincluding from colorectal cancer, lung cancer, breast cancer,endometrial and ovarian cancer, gastric cancer, renal cell cancer,urothelial cancer, hepatocellular cancer, oral squamous cancer, thyroidtumors and glioblastomas. In another embodiment, the multispecificantibody is for use in the treatment of adenoid cystic carcinoma of thehead and neck.

Similarly, the invention relates to a method of treating a diseasecomprising administration of a multispecific antibody according to theinvention as described herein to a human subject in need thereof. In oneembodiment, the disease is cancer, such as prostate cancer, e.g.metastatic or non-metastatic prostate cancer.

In some embodiments, the antibody is administered as monotherapy.However, antibodies of the present invention may also be administered incombination therapy, i.e., combined with other therapeutic agentsrelevant for the disease or condition to be treated.

“Treatment” or “treating” refers to the administration of an effectiveamount of an antibody according to the present invention with thepurpose of easing, ameliorating, arresting, eradicating (curing) orpreventing symptoms or disease states. An “effective amount” refers toan amount effective, at dosages and for periods of time necessary, toachieve a desired therapeutic result. An effective amount of apolypeptide, such as an antibody, may vary according to factors such asthe disease stage, age, sex, and weight of the individual, and theability of the antibody to elicit a desired response in the individual.An effective amount is also one in which any toxic or detrimentaleffects of the antibody are outweighed by the therapeutically beneficialeffects. An exemplary, non-limiting range for an effective amount of anantibody of the present invention is about 0.1 μg/kg to 100 mg/kg, suchas about 1 μg/kg to 50 mg/kg, for example about 0.01 to 20 mg/kg, suchas about 0.1 to 10 mg/kg, for instance about 0.5, about 0.3, about 1,about 3, about 5, or about 8 mg/kg. Administration may be carried out byany suitable route, but will typically be parenteral, such asintravenous, intramuscular or subcutaneous.

Multispecific antibodies of the invention are typically producedrecombinantly, i.e. by expression of nucleic acid constructs encodingthe antibodies in suitable host cells, followed by purification of theproduced recombinant antibody from the cell culture. Nucleic acidconstructs can be produced by standard molecular biological techniqueswell-known in the art. The constructs are typically introduced into thehost cell using an expression vector. Suitable nucleic acid constructsand expression vectors are known in the art. Host cells suitable for therecombinant expression of antibodies are well-known in the art, andinclude CHO, HEK-293, Expi293F, PER-C6, NS/0 and Sp2/0 cells.

Accordingly, in a further aspect, the invention relates to a nucleicacid construct encoding a multispecific antibody according to theinvention. In one embodiment, the construct is a DNA construct. Inanother embodiment, the construct is an RNA construct.

In a further aspect, the invention relates to an expression vectorcomprising a nucleic acid construct encoding a multispecific antibodyaccording to the invention.

In a further aspect, the invention relates to a host cell, i.e. arecombinant host cell, such as a mammalian host cell, preferably a CHOcell comprising one or more nucleic acid constructs encoding amultispecific antibody according to the invention or an expressionvector comprising a nucleic acid construct encoding a multispecificantibody according to the invention.

Accordingly, in a further aspect, the invention relates to production ofa multispecific antibody of the invention, preferably a multispecificantibody of the invention comprising an Fc region, by (co-)expression ofone or more nucleic acid constructs encoding the multispecific antibodyin a suitable host cell, such as a mammalian host cell, e.g. a CHO cell,followed by purification of the produced recombinant antibody from thecell culture or the supernatant after removal of the cells.

TABLE 1 Sequence listing. SEQ ID. code Description Sequence 1 LV1044-VHH EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMH JVZ007WVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTIS RDNAKNTLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVSS 2 LV1050 VHH EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAGTTDYADSVKGRFTI SRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGQGTQVTVSS 3 LV1051 VHH EVQLVESGGGSVQPGGSLRLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAGTTDYADSVKGRFT ISRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGLGTQVTVSS 4 5C8 VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAMGWFRQAPGKEREFVAAISWSGGSTSYADSVKGRF TISRDNAKNTVYLQMNSPKPEDTAIYYCAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVSS 5 5C8var1 VHHEVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAM SWFRQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGA DYGFGRLGIRGYEYDYWGQGTQVTVSS 6 linkerGGGGS 7 LV1044- Bispecific Ab EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMH 5C8WVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTIS RDNAKNTLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSSGGGGSEVQLVESGGGLVQAGGSLRLSC AASGRPFSNYAMGWFRQAPGKEREFVAAISWSGGSTSYADSVKGRFTISRDNAKNTVYLQMNSPKPEDT AIYYCAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVSS 8 LV1050- Bispecific Ab EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMH5C8var1 WVRQAPGKGLEWVSTINPAGTTDYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGQ GTQVTVSSGGGGSEVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWFRQAPGKEREFVSAISWSG GSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGADYGFGRLGIRGYEYDYWGQGT QVTVSSAAAEPEA 9 LV1051- Bispecific AbEVQLVESGGGSVQPGGSLRLSCAASRFMISEYSM 5C8var1HWVRQAPGKGLEWVSTINPAGTTDYADSVKGRFT ISRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGLGTQVTVSSGGGGSEVQLLESGGGSVQPGGSLRLS CAASGRPFSNYAMSWFRQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVSSAAAEPEA 10 Modified AAASDKTHTCPPCP hinge region sequence 11 wtIgG1CH2 and CH3 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV (G1m17,SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY G1m(z)RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT allotype)ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG 12 IgG1 Heavy chainAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLM L234F regionISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN L235E constantAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK T366W variantCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 13 IgG1Heavy chain AAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLM L234F constantISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN L235E regionAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK T366S variantCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD L368AELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN Y407VYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 14 LV1050 CDR1RFMISEYSMH 15 LV1050 CDR2 TINPAGTTDYADSVKG 16 LV1050 CDR3 DGYGY 175C8var1 CDR1 NYAMS 18 5C8var1 CDR2 AISWSGGSTSYADSVKG 19 5C8var1 CDR3QFSGADYGFGRLGIRGYEYDY 20 6H4 VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYGMGWFRQAPGKKREFVAGISWSGGSTDYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFSGAETAYYPSDDYDYWGQGTQVTVSS 21 6H4 CDR1 GRPFSNYGMG 22 6H4 CDR2GISWSGGSTDYADSVKG 23 6H4 CDR3 VFSGAETAYYPSDDYDY 24 PSMAMWNLLHETDSAVATARRPRWLCAGALVLAGGFFLL GFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNY ARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLP GGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSW RGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDP QSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEG NYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLG IASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDY AVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMF LERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQA AAETLSEVA 25 5C8var1EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAM Fc SWFRQAPGKEREFVSAISWSGGSTSYADSVKGRFL234F TISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGA L235EDYGFGRLGIRGYEYDYWGQGTQVTVSSAAASDKT T366WHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 26 LV1050 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMH FcWVRQAPGKGLEWVSTINPAGTTDYADSVKGRFTI L234FSRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGQ L235EGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLF T366SPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY L368AVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD Y407VWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG 275C8var1 EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAM FcSWFRQAPGKEREFVSAISWSGGSTSYADSVKGRF L234FTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGA L235EDYGFGRLGIRGYEYDYWGQGTQVTVSSAAASDKT T366WHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 28 LV1050 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMH FcWVRQAPGKGLEWVSTINPAGTTDYADSVKGRFTI L234FSRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGQ L235EGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLF T366SPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY L368AVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD Y407VWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 29LV1050- EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMH 5C8var1WVRQAPGKGLEWVSTINPAGTTDYADSVKGRFTI -no-c-SRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGQ tagGTQVTVSSGGGGSEVQLLESGGGSVQPGGSLRLS CAASGRPFSNYAMSWFRQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVSS 30 PSMA MARPLCTLLLLMATLAGALAGSHHHHHHGSKSSN varEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLA GTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERD MKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILN LNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVG PGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIV RSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPL MYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTK NWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADK IYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGL PDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA 31 huVdelta2MQRISSLIHLSLFWAGVMSAIELVPEHQTVPVSIGV ECD-PATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYRE FcKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEG (hole)-SYYCACDTLGMGGEYTDKLIFGKGTRVTVEPRSQP HisHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQH DNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTAAASDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGHHHHHH 32 huVgamma9MLSLLHASTLAVLGALCVYGAGHLEQPQISSTKTLS ECD-FcKTARLECVVSGITISATSVYWYRERPGEVIQFLVSI (knob)-SYDGTVRKESGIPSGKFEVDRIPETSTSTLTIHNVE C-tagKQDIATYYCALWEAQQELGKKIKVFGPGTKLIITDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFP DVIKIHWEEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIK TDVITMDPKDNCSKDANDTLLLQLTNTSAAAASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAAAEPEA

EXAMPLES Example 1: PBMC Isolation and Generation of Human Donor-DerivedVγ9Vβ2-T Cell Cultures

Whole blood was collected from healthy donor volunteers. Alternatively,buffy coats were obtained from blood supply service Sanquin and used forisolation of peripheral blood mononuclear cells (PBMC). PBMC wereisolated using Lymphoprep™ density gradient centrifugation. Vγ9Vδ2-Tcells were then isolated from healthy donor PBMC by magnetic-activatedcell sorting (MACS) using a FITC-labeled anti-TCR Vδ2 mouse monoclonalantibody (Mab) in combination with goat anti-mouse IgG microbeads.Purified Vγ9Vδ2-T cells were stimulated every seven days with a feedercell mix consisting of irradiated PBMC from two healthy donors and anEpstein Barr Virus transformed B cell line (JY) resuspended in RoswellPark Memorial Institute (RPMI) medium supplemented with 10 IU/mL IL-7,10 ng/mL IL-15 and 50 ng/ml PHA. Expanded Vγ9Vδ2-T cell cultures werealways tested for purity before being used for experiments and alwaysfound to be >95% Vγ9 and Vδ2 double positive.

Example 2: Humanization of the Anti-PSMA VHH JVZ-007 and Anti-Vγ9Vβ2 VHH5C8

The amino acid sequence of the camelid-derived anti-PSMA VHH JVZ-007(LV1044; SEQ ID NO:1) (J Nucl Med. 2015 July; 56(7):1094-9, supplementaldata) was aligned to the human V gene database and the closest humangermline match was found to be IGHV3-74*01. Based on sequencedifferences in the framework regions between the human and llama-derivedsequence, two humanized variants (SEQ ID NO:2-3) were designed, seealignment in FIG. 1 .

JVZ-007 and these two mutants were then combined with anti-Vγ9Vδ2 VHH5C8 (a Vδ2 binding antibody described in WO2015156673) (SEQ ID NO:4) or5C8var1, a humanized variant of 5C8 (SEQ ID NO:5), in bispecific VHHformat: anti-PSMA VHH-linker-anti Vγ9Vδ2 VHH. The sequence of the linkerwas GGGGS (SEQ ID NO:6). A C-terminal C-tag (EPEA sequence) preceded bythree alanine residues was in some constructs added for purification anddetection purposes. The sequences of the resulting constructs are setforth in SEQ ID NO:7-9.

Example 3: Design of a Half-Life Extended Version of the Bispecific VHHFormat

A half-life extended version of the bispecific VHH format was designed,based on an Fc-containing format. VHH sequences were coupled to aslightly modified human IgG1 hinge region: Amino acid residues 216-230(EU numbering) were changed to AAASDKTHTCPPCP (SEQ ID NO: 10). Thisomits the cysteine that normally bridges to the other heavy chain andreplaces the ‘EPK’ upper hinge sequence with three alanines. Thismodified hinge was coupled to an IgG1 (G1m17, G1m(z) allotypesequence)-CH2 and -CH3 sequences (SEQ ID NO:11). The CH2 sequence wasmodified to contain Fc silencing mutations (L234F, L235E) and the CH3sequence was modified to either contain a ‘knob’ mutation (T366W) (SEQID NO:12), or three mutations that create a ‘hole’ (T366S, L368A andY407V) (SEQ ID NO: 13). This knob-into-hole (KiH) technology induces thepreferential hetero-dimerization of the two chains. The resultingconstruct is schematically depicted in FIG. 2 .

Example 4: Cloning, Expression and Purification of Bispecific VHHMolecules and Fc-Containing Constructs

Amino acid sequences of bispecific VHH molecules were reverse-translatedto cDNA and then codon-optimized for expression in human cells.Regulatory elements were added: an N-terminal Kozak sequence andC-terminal stop codon (including BamH1 and Not1 restriction sites forcloning) and the cDNA was made as a synthetic gene. cDNAs were clonedinto a suitable vector and their sequences were verified. Expression ofthe proteins was performed by transient transfection of the resultingplasmids in HEK293_E cells. Proteins were purified from the culturesupernatant by means of protein-A affinity chromatography andpreparative size-exclusion chromatography.

The amino acid sequences of the two protein chains of Fc-containingconstructs were reverse-translated into the encoding cDNA, necessaryregulatory elements were added (Kozak sequence, stop codon and cloningsites BamH1 and Not1) and the cDNAs were codon-optimized for expression.The cDNAs were made by synthetic gene synthesis and expression plasmidsencoding either of the two protein chains were made by cloning the cDNAsseparately into a suitable vector. The resulting plasmids weresequence-verified and then used to transfect CHO cells grown insuspension using different ratios of the two plasmids (1:2, 1:1 and2:1). Secreted proteins were purified from the culture supernatant usingprotein-A affinity chromatography and were buffer-exchanged to PBS.Proteins were further purified by preparative size-exclusionchromatography.

Example 5: Testing of Humanization Variants of Anti-PSMA VHH JVZ-007

Purified bispecific VHHs containing the humanization variants weretested for their ability to induce target-dependent T cell activation ina 4-hour degranulation assay. Vγ9Vδ2 T cells were isolated from PBMC bymeans of magnetic activated cell sorting (MACS) and expanded asdescribed in Example 1. Expanded Vγ9Vδ2 T cells used for experimentswere always checked for purity and found to be >95% double positive forVγ9- and Vδ2 staining in FACS. Purified, expanded Vγ9Vδ2 T cells wereincubated with the same number of PSMA-positive LNCaP (ATCC, cat. Nr.CRL-1740) target cells (effector: target (E:T) cell ratio of 1:1) and aconcentration range of bispecific construct. After 4 hours ofincubation, the percentage of T cells expressing CD107a was determinedby staining in FACS.

FIG. 3 shows that the humanization variants of the PSMA VHH JVZ-007showed a strong potency in inducing Vγ9Vδ2 T cell degranulationdependent on the antigen-positive cell line LNCaP. However, a singlehigh concentration of bispecific construct did not cause significantactivation of T cells when the antigen-negative cell line PC-3 was used.

Example 6: Cytotoxicity Mediated by Bispecific VHH and Fc-ContainingCounterpart

The two VHH sequences of bispecific VHH LV1050-5C8var1 were re-formattedinto a half-life extended molecule containing a human IgG1 Fc asdescribed above, resulting in the constructs set forth in SEQ ID NO:25and SEQ ID NO:26 (5C8var1-Fc and LV1050-Fc).

Both the bispecific VHH construct LV1050-5C8var1 and the Fc-containingcounterpart 5C8var1-Fc x LV1050-Fc (also termed LV1050-Fc x 5C8var1herein) were tested for their ability to induce cytotoxicity of LNCaPcells through activation of Vγ9Vδ2 T cells.

FIG. 4 shows that both the bispecific VHH, as well as the Fc-containingcounterpart containing the same VHH sequences were able to induce 100%tumor cell lysis after 24 hours in an E:T ratio of 1:1. The bispecificVHH LV1044-5C8 containing the original anti-PSMA VHH JVZ-007 was aspotent as the humanized version LV1050-5C8var1 and both of these wereslightly more potent than the Fc-containing counterpart 5C8var1-Fc xLV1050-Fc. EC50 values found were 2.2 pM and 1.6 pM for LV1050-5C8var1and LV1044-5C8 respectively and 10.5 pM for 5C8var1-Fc x LV1050-Fc. Forsubsequent experiments, a C-terminal lysine was added for expressionpurposes to both constructs encoding Fc-containing chains resulting inthe coding sequences set forth in SEQ ID NO:27 and SEQ ID NO:28.Analysis of the produced polypeptides revealed that the C-terminallysines were clipped off (data not shown), thus resulting in identicalpolypeptides to those produced from constructs not encoding a C-terminallysine.

Example 7: Binding of LV1050-Fc x 5C8var1-Fc to Target Positive Cells inFACS

Bispecific Fc-containing construct 5C8var1-Fc x LV1050-Fc cloned,expressed and purified as described above. To test the binding of themolecule to PSMA, a concentration range of antibody was tested forbinding to the PSMA positive prostate cancer cell line LNCaP and to theantigen negative cell line PC-3. Detection was performed with apolyclonal anti-human IgG antibody.

To test the binding of LV1050-Fc x 5C8var1-Fc to Vγ9Vδ2 T cells, aconcentration range of the compound (100 nM and a half-log dilutionthereof) was tested for binding in FACS staining using detection withtwo different monoclonal antibodies directed towards VHH (45H8 and96A3F5; Genscript, cat. Nrs CP001/18L001614 and A01994 respectively).

FIG. 5 shows that the molecule binds specifically to PSMA (left panel),as it did not measurably stain the PSMA-negative cell line PC-3, butshowed strong binding to LNCaP cells. The EC50 for binding was measuredto be 7.3 nM. The affinity for the Vγ9Vδ2 TCR was measured to be 1.7 nMby FACS (right panel).

Example 8: Lead Bispecific Antibody LV1050-Fc x 5C8var1-Fc InducesPotent Target-Dependent T Cell Activation and Target Cell Lysis

To measure the potency of the bispecific T cell engager, target-positiveLNCaP cells were incubated with a concentration range of the moleculeand a fixed number of Vγ9Vδ2 T cells (effector:target ratio of 1:1). Tcell activation was then measured by staining in FACS for CD107aexpression on the T cells after four hours of incubation. Cytotoxicitywas measured after 24 hours by measuring the number of viable cells inFACS (7AAD staining). FIG. 6 shows that LV1050-Fc x 5C8var1-Fc inducespotent T cell activation as witnessed by CD107a expression. In addition,this resulted in a strong cytotoxic effect on the LNCaP target cells.The EC50 values for cytotoxicity that were found were virtually the samefor both bispecific VHH molecules tested (1.9 pM for LV1044-5C8(produced without AAAEPEA tag in this experiment) and LV1050-5C8var1)and slightly higher for LV1050-Fc x 5C8var1-Fc (9.4 pM).

Example 9: Frequency Vγ9Vβ2-T Cells and Expression of Ligands inPatient-Derived Tumor and Normal Tissue

Prostate tumor tissue was obtained after radical prostatectomy frompatients with non-metastatic prostate cancer. Both macroscopicallynormal tissue, as well as tumor tissue was analyzed. Tissue was cut intosmall pieces with a surgical blade and resuspended in dissociationmedium composed of Iscove Modified Dulbecco Medium (IMDM) supplementedwith 0.1% DNAse I (Roche), 0.14% Collagenase A, 100 IU/mL sodiumpenicillin/100 μg/mL streptomycin sulphate/2.0 mM L glutamine and 5%FCS. The tissue pieces were transferred to a sterile flask and incubatedon a magnetic stirrer for 45 minutes at 37 degrees. After thisincubation, the cell suspension was run through a 100 μM cell strainer.Tumor tissue was dissociated three times and normal tissue twice intotal, after which cells were washed and prepared for viable cell countusing trypan blue exclusion. Dissociated tumor- and normal tissues wereanalyzed for the presence of Vγ9Vδ2-T cells using staining in FACS withAF700 labeled anti-CD45 Mab, PerCP-Cy5.5 labeled anti-CD3 mAb, APClabeled anti-TCR Vγ9 mAb and BV711 labeled anti-TCR Vδ2 mAb. Expressionof the targets EpCAM, PSMA and CD277 on tumor- and normal cells wasdetermined using BV421 labeled anti-EpCAM Mab, FITC labeled anti-PSMAMab and PE labeled anti-CD277 Mab.

FIG. 7 shows that there is a significant difference in both PSMA− aswell as CD277 expression between tumor and normal tissue. Resectedtissue was macroscopically examined and then defined as being tumor ornormal tissue. This was then further corroborated by EpCAM positivestaining for tumor in FACS, which correlated with PSMA expression. PSMAwas almost absent from normal tissue and significantly expressed ondissociated tumour cells. CD277 (BTN3A) expression was also more highlyexpressed on tumour, although the difference lacked statisticalsignificance. In contrast, the frequency of Vγ9Vδ2-T cells in bothtumor- and normal tissue was largely comparable, with normal tissuehaving a slightly higher percentage.

Example 10: Functional Analysis of LV1044-5C8 Using Patient-DerivedTarget Cells

To determine the potential of the bispecific constructs to mediatecytotoxicity Vγ9Vδ2-T cells against patient-derived target cells,dissociated tumor- and normal cell suspensions were incubated for 24hours at 37° C. with or without 50 nM of compound and Vγ9Vδ2-T cellcultures in a 1:1 effector:target (E:T) ratio. The number of livingtarget cells was determined using the life-death marker 7AAD and 123count eBeads.

FIG. 8 shows that Vγ9Vδ2 T cells were capable of inducing tumor cellkill of patient-derived tumor cells in a target- and Vγ9Vδ2 T-celldependent manner. Normal tissue that was largely devoid of PSMAexpression was not affected by Vγ9Vδ2 T cells, even in the presence of50 nM of bispecific antibody. However, PSMA-positive tumor cells weresignificantly killed after 24 hours, but only in the presence of botheffector cells and bispecific antibody.

Example 11: Affinity Determination LV1050-Fc x 5C8var1-Fc for Human PSMAand Vγ9Vβ2 TCR Using Biolayer Interferometry

The affinities of LV1050-Fc x 5C8var1-Fc for recombinant human PSMA andhuman Vγ9Vδ2 TCR were determined using biolayer interferometry (BLI). Asligand, 12.5 μg/mL biotinylated hPSMA or 5 μg/mL hVγ9Vδ2-Fc (consistingof SEQ ID NO:31 and 32) were loaded onto streptavidin biosensors. Asanalyte, two-fold serial dilutions of LV1050-Fc x 5C8var1-Fc were used:ranging from 3.125 to 200 nM in combination with ligand hPSMA; rangingfrom 0.03125 to 20 nM in combination with ligand hVγ9Vδ2-Fc. As shown inthe Table 2, LV1050-Fc x 5C8var1-Fc binds with a K_(D) value of 32±1.2nM to human PSMA, and with a K_(D) of 0.64±0.16 nM to the human Vγ9Vδ2TCR.

TABLE 2 Affinity determination LV1050-Fc × 5C8var1-Fc for human PSMA andVγ9Vδ2 TCR using biolayer interferometry K_(D) (M) k_(on) (1/Ms) k_(dis)(1/s) Ligand mean SD k_(on) (1/Ms) SD k_(dis) (1/s) SD PSMA (n = 3)3.16E−08 1.24E−09 3.07E+05 7.41E+03 9.69E−03 4.73E−04 Vγ9Vδ2 6.35E−101.60E−10 2.40E+05 8.64E+03 1.51E−04 3.40E−05 TCR (n = 4)

Example 12: Epitope Mapping

To determine the epitope on PSMA bound by the VHH incorporated inLV1050-Fc x 5C8var1-Fc, LV1050-5C8var1-no-c-tag (SEQ ID NO:29)(containing the identical PSMA binding VHH domain as LV1050-Fc x5C8var1-Fc) was used in the epitope mapping technology developed byCovalX AG (Pimenova et al. (2008) J Mass Spectrom 43:185). In short, arecombinant, PSMA protein (SEQ ID NO: 30) was allowed to bind toLV1050-5C8var1-no-c-tag, cross-linked, exposed to different proteasesand the resulting peptides, cross-linked or not, were analyzed byhigh-resolution mass spectrometry.

The results demonstrated the VHH to bind a conformational epitope whichis shown in FIG. 9 . The following residues were found to have a directinteraction with the antibody: R149, S156, R163, S276, R279, S281, K283,H577, 5590, K688, R689 and Y692. These residues correspond to residuesR190, S197, R204, S317, R320, S322, K324, H618, S631, K729, R730 andY733 in UniProt sequence Q04609-1. None of the rare single nucleotidepolymorphisms (SNPs) that have been identified in the FOLH1 gene werepresent in the epitope, suggesting LV1050-5C8var1-no-c-tag, andtherefore LV1050-Fc x 5C8var1-Fc, to be capable of binding all relevantidentified SNP-variants of the target.

Example 13: Further Functional Analysis of LV1050-Fc x 5C8var1-Fc UsingTumor Derived Cell Lines

The ability of LV1050-Fc x 5C8var1-Fc to induce PSMA-dependent Vγ9Vδ2-Tcell activation was determined in a 4-hour in vitro assay usingexpression of the degranulation marker CD107a (LAMP-1) as read-out.Expanded Vγ9Vδ2 T cells isolated from PBMCs from healthy donors werecultured with PSMA-expressing prostate-derived cancer cell lines LNCaP,VCaP or 22Rv1 in a 1:1 effector: target cell ratio, in the absence orpresence of different concentrations of LV1050-Fc x 5C8var1-Fc (rangingfrom 10 fM to 3.16 nM). Cells were harvested and the cell surfaceexpression of CD107a was determined by flow cytometry. In the presenceof all PSMA expressing tumor cell lines, LV1050-Fc x 5C8var1-Fc inducedvery potent Vγ9Vδ2-T cell activation (FIG. 10 ) with EC₅₀ values in pMrange (Table 3).

TABLE 3 EC₅₀ values of Vγ9Vδ2-T cell degranulation and Vγ9Vδ2-T cellmediated tumor cell cytotoxicity with LV1050-Fc × 5C8var1-Fc in in vitroassays using Vγ9Vδ2-T cells and PSMA positive tumor cell lines TargetTested Mean EC₅₀ Number of Effector cell range E:T (nM) donors (n) andcells line (nM) ratio (±SD) experiments Degranulation after 4 hoursVγ9Vδ2-T LNCaP 3.2- 1:1 0.016 n = 10 (10 cells 0.000010 (0.0049) donorsin 5 exp) Vγ9Vδ2-T 22Rv1 3.2- 1:1 0.029 n = 4 (4 cells 0.000032 (0.0049)donors in 2 exp) Vγ9Vδ2-T VCaP 3.2- 1:1 0.028 n = 4 (4 cells 0.000032(0.0064) donors in 2 exp) Cytotoxicity after 24 hours Vγ9Vδ2-T LNCaP3.2- 1:1 0.017 n = 10 (10 cells 0.000010 (0.013) donors in 7 exp, 4donors tested twice) Vγ9Vδ2-T 22Rv1 3.2- 1:1 0.013 n = 4 (4 cells0.000032 (0.0069) donors in 2 exp) Vγ9Vδ2-T VCaP 3.2- 1:1 0.015 n = 3 (3cells 0.000032 (0.011) donors in 2 exp)

Next, the capacity of LV1050-Fc x 5C8var1-Fc to induce target-dependent,Vγ9Vδ2-T cell-mediated tumor cell killing was determined in the sameco-cultures at 24 hours, using a luminescence assay to quantify therelease of an intracellular protease from dying/dead tumor cells(CytoTox-Glo™ Cytotoxicity Assay, Promega). LV1050-Fc x 5C8var1-Fcinduced strong, target-dependent and Vγ9Vδ2-T cell-mediated cytotoxicityof LNCaP, VCaP and 22Rv1 cells (FIG. 11B) with EC₅₀ values in the samerange as for degranulation (Table 3).

In addition, Vγ9Vδ2-T cells were not activated by LV1050-Fc x 5C8var1-Fcalone (in the absence of tumor cells), neither was lysis observed oftumor cells in which PSMA expression was abrogated (LNCaP.koPSMA)(FIG.11A).

To study the effect of different E:T ratios on the potency of LV1050-Fcx 5C8var1-Fc (concentration range tested: 10 fM to 3.2 nM), the numberof target LNCaP cells was kept constant (i.e. 50,000) in a 24-hourcytotoxicity assay, while the number of effector Vγ9Vδ2-T cells wasvaried to obtain different E:T ratios (1:1, 1:10 and 1:100). As shown inTable 4, mean EC₅₀ values for Vγ9Vδ2-T cell-mediated cytotoxicityinduced by LV1050-Fc x 5C8var1-Fc were comparable between E:T ratio 1:1and 1:10 (17±13 pM and 9.9±6.5 pM, respectively), while at the E:T ratioof 1:100, the level of cytotoxicity observed was too low to accuratelycalculate an EC₅₀ value. Even though the potency (EC₅₀) of LV1050-Fc x5C8var1-Fc in the assay was not affected, the maximum percentage oflysis observed was significantly lowered in the E:T ratio 1:10 comparedto 1:1 E:T ratio (data not shown). Thus, the potency (i.e. EC₅₀ measuredin the assay) of LV1050-Fc x 5C8var1-Fc is not strongly affected byvarying E:T ratios.

TABLE 4 EC₂₀, EC₅₀ and EC₉₀ values of Vγ9Vδ2-T cell cytotoxicitymediated by LV1050-Fc × 5C8var1-Fc using different E:T ratios. Mean MeanMean Number of E:T EC20 ± EC50 EC90 ± donors (n) ratio SD (pM) SD (pM)SD (pM) and experiments 1:1  4.3 ± 3.0 17 ± 13 79 ± 48 n = 10 (10 donorsin 7 expts, 4 donors tested twice) 1:10 3.6 ± 1.3 9.9 ± 6.5 60 ± 59 n =4 (4 donors in 2 expts)

Example 14: Functional Analysis of LV1050-Fc x 5C8var1-Fc UsingPatient-Derived Target Cells

Patient-derived non-metastatic prostate cancer tissue was obtained andnormal (non-malignant) and tumor tissue were processed as described inExample 9.

As a measure of activation of Vγ9Vδ2-T cells, upregulation of thedegranulation marker CD107a (LAMP-1; detected using PE-labeledanti-human CD107a, Thermofisher) on Vγ9Vδ2-T cells present in thedissociated prostate samples (0.5-1×10⁵ dissociated cells from normal(non-malignant) or primary tumor tissue) was determined in the presenceor absence of 50 nM LV1050-Fc x 5C8var1-Fc. After 4 hours, degranulationof Vγ9Vδ2-T cells was measured by CD107a expression and analyzed by aflow cytometry-based assay (determination of the percentage ofEpCAM−/CD45+/CD3+/Vγ9+/Vδ2+/CD107a+ cells).

Also, the ability of LV1050-Fc x 5C8var1-Fc to trigger degranulation ofVγ9Vδ2-T cells in autologous patient PMBC cultured in the presence ofprostate tumor or normal (non-malignant) tissue was determined. To thisaim, the same method as above was used, with the exception that hereautologous PBMC were added at an effector to target (E:T; PBMC:prostatecell) ratio of 10:1 and incubated for 24 hours.

In dissociated prostate cancer tissue, but not in non-malignant prostatetissue, LV1050-Fc x 5C8var1-Fc induced a statistically significantincrease in degranulation of tissue infiltrating Vγ9Vδ2-T cells. Asignificantly higher percentage of CD107a-expressing Vγ9Vδ2-T cells wasobserved compared to incubation of tissue cells without the bispecificantibody (FIG. 12A). In normal tissue, LV1050-Fc x 5C8var1-Fc did notinduce degranulation of tissue infiltrating Vγ9Vδ2-T cells (FIG. 12B).

In addition, the ability of LV1050-Fc x 5C8var1-Fc to activate Vγ9Vδ2-Tcells in autologous PMBC upon co-culture with patient prostate tumor ornormal (non-malignant) tissue was determined. Co-culture in the presenceof LV1050-Fc x 5C8var1-Fc resulted in a significantly higher percentageof CD107a-expressing Vγ9Vδ2-T cells compared to incubation of tumortissue cells and autologous PMBC alone (FIG. 13A). No activation ofVγ9Vδ2-T cells in autologous PBMC was observed when LV1050-Fc x5C8var1-Fc was added to cultures of autologous PMBC and normal(non-malignant) prostate tissue (FIG. 13B).

The specific cytotoxicity induced by LV1050-Fc x 5C8var1-Fc was testedin co-cultures of prostate tumor cells or normal (non-malignant)prostate cells and autologous PBMC as described in Example 10, exceptthat an E:T ratio of 10:1 was used. LV1050-Fc x 5C8var1-Fc inducedstatistically significant lysis of tumor cells in the presence ofautologous PBMC, whereas normal (non-malignant) prostate cells were notlysed (FIG. 14 ).

Example 15: In Vivo Therapeutic Efficacy of LV1050-Fc x 5C8var1-Fc

Immunodeficient NCG mice were subcutaneously inoculated with 5×10⁶ 22Rv1cells mixed with human PBMC in a 2:1 ratio (22Rv1:PBMC). PBMC from 2donors showing different frequencies of Vγ9Vδ2-T cells in CD3+ T cells,donor #1 (21.9% Vγ9Vδ2-T cells) and donor #2 (8.8% Vγ9Vδ2-T cells), wereused in this study. LV1050-Fc x 5C8var1-Fc (0.2 mg/kg or 2 mg/kg) orphosphate buffered saline (PBS) was administered IV every week (days 0,7, 14 and 21) starting at the day of tumor cell- and PBMC inoculation.Tumor sizes were measured in two dimensions twice a week using acaliper. Mice were sacrificed when the mean tumor volume of a groupexceeded 2,000 mm³.

For mice injected with PBMC from donor #1, LV1050-Fc x 5C8var1-Fcadministration at 0.2 or 2 mg/kg resulted in statistically significanttumor growth inhibition (TGI) values of 91% and 78%, respectively, onday 34 (when mice in the control (PBS-treated) group were sacrificed:FIG. 15A). For mice injected with PBMC from donor #2, the TGI valuesobserved on day 41 (when the mice in the corresponding PBS-treated groupwere sacrificed) were 23% and 52% for mice treated with 0.2 and 2 mg/kgof LV1050-Fc x 5C8var1-Fc, respectively (FIG. 15B). For donor #2,statistical significance in TGI was found for the highest dose of thecompound administered.

Example 16: Whole Blood Cytokine Release Evaluation

An in vitro cytokine release assay was performed using a solution phaseassay employing fresh whole blood from 30 healthy donors. Differentconcentrations of LV1050-Fc x 5C8var1-Fc (ranging from 280 to 8.75 nM)were incubated for 24 hours with whole blood and the levels of sevencytokines (IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and tumor necrosisfactor (TNF)α) in plasma were measured using an immunoassay. Erbitux®(cetuximab) and Campath® (alemtuzumab) were included in the assay ascontrol compounds inducing low and high cytokine release in the clinic,respectively. Staphylococcal enterotoxin B (SEB) was used as a positiveassay control for the release of all the cytokines tested.

A summary of the results is shown in Table 5. LV1050-Fc x 5C8var1-Fconly induced the release of IL-8 and IFN-γ. The LV1050-Fc x5C8var1-Fc-induced release of IL-8 was comparable to that induced byErbitux®, an antibody that is known not to induce cytokine releasesyndrome (CRS) (or is known to induce low levels of cytokines) inpatients. LV1050-Fc x 5C8var1-Fc induced an IFN-γ release that wasslightly higher when compared to Erbitux®, but the highest IFN-γ releaseobserved was much lower than that induced by Campath®, an antibodyclinically associated with CRS. Importantly, LV1050-Fc x 5C8var1-Fc didnot induce any IL-6 release, a prominent cytokine in CRS (Tanaka et al(2016) Immunotherapy 8: 959).

In an in vitro cytokine release assay where LV1050-Fc x 5C8var1-Fc wascoated to high-binding 96 wells plates prior to incubation with freshwhole blood from 10 healthy donors, similar results were obtained. Here,LV1050-Fc x 5C8var1-Fc induced very low (median) levels of IL-6 andIL-8, which were comparable to the low response comparator, Erbitux® andno TNFα release was observed. LV1050-Fc x 5C8var1-Fc induced higher(median) levels of IFN γ compared with Erbitux® at each concentration ofthe compound tested, yet median levels were substantially lower thanthat induced by Campath®.

TABLE 5 Cytokine release profile (pg/mL) induced by LV1050-Fc ×5C8var1-Fc in fresh blood samples of healthy donors. Test Article IL-2IL-4 IL-6 IL-8 IL-10 IFN-γ TNFα PBS (negative control) 0 0 0 0 0 0 0 SEB(positive control) 24,470.9 53.3 5,613.2 8,936.3 1,010.3 61,792.24,302.2 Erbitux ® 280 nM 0 0 0 15.1 0 0 0 140 nM 0 0 0 24.8 0 1.1 0 70nM 0 0 4.7 15.6 0 0.5 0 35 nM 0 0 17.4 20.0 0 0 0 17.5 nM 0 0 46.0 16.60 0 0 8.75 nM 0 0 6.2 15.4 0 3.6 0 Campath ® 280 nM 0 0 1,732.1 578.20.2 3,409.9 18.1 140 nM 0 0 1,360.3 392.7 0 4,029.0 12.9 70 nM 0 0 896.5326.5 0 4,732.9 8.3 35 nM 0 0 714.9 255.5 0 5,313.4 14.5 17.5 nM 0 0422.4 195.1 0 4,353.3 16.9 8.75 nM 0 0 400.9 180.5 0 4,932.2 20.6LV1050-Fc × 280 nM 0 0 0 35.4 0 9.4 0 5C8var1-Fc 140 nM 0 0 0 25.4 024.4 0 70 nM 0 0 0 24.2 0 37.9 0 35 nM 0 0 0 20.3 0 49.6 0 17.5 nM 0 0 028.2 0 50.0 0 8.75 nM* 0 0 0 19.2 0 81.4 0

Median cytokine levels (pg/mL) for each drug/dose combination measuredin fresh blood samples of 30 healthy donors by an in vitro cytokinerelease assay.

Example 17: Pharmacokinetics

LV1050-Fc x 5C8var1-Fc contains a human Fc domain, which is expected toextend the in vivo half-life of the compound by binding to the humanFcRn receptor. To verify this, LV1050-Fc x 5C8var1-Fc wasco-administered with a human IgG control in three single IV doses of 2mg/kg, 5 mg/kg and 10 mg/kg in human FcRn Tg32 SCID mice (Jackson labs:JAX). Blood samples were collected at different time points (5 m, 8 h,1, 3, 7, 10, 14, 17, 21 and 28 days) after administration andconcentrations of LV1050-Fc x 5C8var1-Fc were assessed using an antigencapture ELISA. The results obtained in this model system show thatLV1050-Fc x 5C8var1-Fc has a half-life in these transgenic mice thatranges between 140 and 172 hours (5.8-7.2 days, FIG. 16 ), comparable tothe half-life that an IgG-based antibody has in this system.

The pharmacokinetics of LV1050-Fc x 5C8var1-Fc were also evaluated innon-human primates (NHP). Three female cynomolgus monkeys were given asingle IV dose of LV1050-Fc x 5C8var1-Fc (0.14, 0.77 and 2.27 mg/kg (1animal per dose)) using a 30-minute IV infusion. The half-life ofLV1050-Fc x 5C8var1-Fc ranged between 150 and 166 hours (6.3-6.9 days)(FIG. 17 ), which is in line with the half-lives of IgG-based humanizedantibodies which do not bind to the cynomolgus ortholog targets (Walkeret al. (2019) PLOS ONE 14: e0217061). A dose-proportional increase inexposure was observed. The pharmacokinetic parameters are provided inTable 6.

TABLE 6 Pharmacokinetic parameters of LV1050-Fc × 5C8var1-Fc incynomolgus monkeys after a single IV dose. Adm. nC_(max) nAUC_(0-∞)Animal dose T_(max) C_(max) (μg/mL)/ T_(1/2) AUC_(0-∞) (μg · h/mL)/ ClVz ID (mg/kg) (hrs) (μg/mL) (mg/kg) (hrs) (μg · h/mL) (mg/kg) (mL/h/kg)(mL/kg) 61 0.14 1 3.17 22.6 162.1 297.9 2127.9 0.47 109.9 62 0.77 1 17.122.2 150.0 1513.2 1965.2 0.51 110.1 63 2.27 1 53.6 23.6 165.9 5548.72444.4 0.41 97.9 CL = clearance; (n)C_(max) = (normalized) maximumconcentration after dosing; (n)AUC_(∞) = (normalized) area under theconcentration versus time curve from time-point t = 0 h to infinity;T_(max) = the time after dosing at which the maximum concentrationreached; t_(1/2) = elimination half-life; V_(z) = apparent volume ofdistribution.

Example 18: Homogeneous Product Irrespective of Transfection Ratio

cDNAs encoding the relevant two antibody heavy chains of LV1050-Fc x5C8var1-Fc were each cloned in expression vectors suitable formanufacturing. Different ratio's, 1:1, 1:1.25, 1:1.5, 1.25:1 and 1.5:1,of expression vectors containing LV1050-Fc or 5C8var1-Fc were used fortransfection of cells suitable for manufacturing. Cells were thenselected for resistance to two different antibiotics and antibodies wereproduced from selected cell pools by means of fed batch production. SizeExclusion High Performance Liquid Chromatography (SE-HPLC) analysis ofthe produced antibodies revealed high product purities (dimer content):between 93.2% and 96.4% of the secreted protein was at the expectedmolecular seize, irrespective of the transfection ratio (main peak areain Table 7). Reverse Phase HPLCd (RP-HPLC) analysis was established tofurther distinguish the monomers (LV1050-Fc and 5C8var1-Fc) andhomodimers (LV1050-Fc x LV1050-Fc and 5C8var1-Fc x 5C8var1-Fc) fromintended LAVA heterodimers (LV1050-Fc x 5C8var1-Fc). All clone poolsamples revealed a high content of intended heterodimer LAVA LV1050-Fc x5C8var1-Fc between 86.1 and 94.9%. Clone pools DGC8-T1P, -T3P and -T5Prevealed an elevated percentage of 5C8var1-Fcx5C8var1-Fc dimer.

TABLE 7 Results of SE-HPLC for clone pools obtained by transfection ofdifferent ratios of expression vectors containing LV1050-Fc or5C8var1-Fc. rel. peak areas [%] Ratio HMW LMW Pool-ID.LV1050-Fc:5c8var1-Fc Species Main species DGC8-T1P 1:1 4.1 95.8 0.0DGC8-T3P   1:1.25 4.5 94.1 1.4 DGC8-T5P  1:1.5 4.3 93.2 2.5 DGC8-T7P1.25:1   3.6 96.4 0.1 DGC8-T9P 1.5:1  3.6 96.3 0.0 HMW—high molecularweight; LMW—low molecular weight

Example 19: Formulation of LV1050-Fc x 5C8var1-Fc

From a selected cell clone, LV1050-Fc x 5C8var1-Fc product was obtainedby means of production in a bioreactor system, and after harvest andpurification formulated at protein concentrations of 0.5 mg/mL and 10mg/mL using four types of formulation buffers:

Buffer Formulation Buffer 1 10 mM Histidine + 280 mM Sucrose + 0.02%Polysorbate 80, pH 6.0 Buffer 2 10 mM Histidine + 280 mM Sucrose + 0.02%Polysorbate 80, pH 6.0 + 1 mM Methionine Buffer 3 10 mM Sodium acetate +280 mM Sucrose + 0.02% Polysorbate 80, pH 5.5 Buffer 4 10 mM Sodiumacetate + 280 mM Sucrose + 0.02% Polysorbate 80, pH 5.5 + 1 mMMethionine

Formulated samples were subjected to several storage conditions (definedbelow), for up to 12 weeks. Additionally, several stress tests wereapplied:

-   -   Storage at 5° C.±3° C.    -   Storage at −80° C.±10° C.    -   Accelerated storage conditions at 25° C.±2° C./60% relative        humidity (RH) t 5%    -   Heat stress at 40° C.±2° C./75% RH±5%    -   Freeze/Thaw cycles: The samples were completely frozen to        −80±10° C. Subsequently, five freeze/thaw cycles have been        performed after allowing samples to completely thaw at room        temperature (15-25° C.).    -   Agitation stress: Samples agitated at 240 rpm for 7 days at room        temperature (EP15° C.-25° C.).    -   Oxidation: The samples were spiked with 0.01% (v/v) hydrogen        peroxide (H₂O₂) and incubated for 7 days at 25° C.±2° C./60%        RH±5%.    -   Photostability: >1.2 million lux hours and >200 watt        hours/square meter of near ultra violet energy were applied at        25° C.±2° C./60% RH±5%.

Initial (=t0) 5° C.±3° C. samples served as a reference. Samples wereanalyzed using the following analytical methods:

Method Assessment Clarity Appearance Color Appearance pH pH A₂₈₀(UV/VIS) Content Stray light Integrity SE-HPLC Integrity Cation-exchange(CIEX)-HPLC Integrity Capillary electrophoresis sodium dodecyl sulfate(CE- Integrity SDS_(non-reduced) CE-SDS (reduced) Integrity Activityassay - PSMA binding ELISA Potency Peptide mapping Identity Differentialscanning Integrity Liquid chromatography-mass spectrometry (LC-MS)Integrity (oxidation)

The results of the stability over time study were highly comparable forClarity, Color, pH, A280 (UV-VIS), Stray light, SE-HPLC, CIEX-HPLC,CE-SDS and PSMA binding, Differential Scanning and Peptide Fingerprintfor storage temperatures at −80° C. t 10° C., 5° C.±3° C. and 25° C.±2°C./60% RH±5%. The results of the measurements indicate that the productwas highly stable with the chosen buffer conditions and only slighteffects were observed over time.

Distinct effects were detected under stress conditions at 40° C.±2°C./75% RH±5% particularly at five weeks, enabling a differentiationbetween the tested buffers and concentrations. The CIEX-HPLC analyses ofsamples kept at 40° C.±2° C./75% RH t 5% and 5 weeks showed that at thistimepoint the content of main variant dropped below 60% for all testedbuffers and product concentrations. In the histidine-based formulation aslightly lower decrease was observed in main variant percentage comparedto the acetate-based formulation.

The CE-SDS results revealed that storage over time at 40° C. resulted ina slight increase of LMW species of the product up to 5%. This effectwas slightly more pronounced for the 10 mg/mL samples in buffer 1 and 3.Nevertheless, the antibody was found very stable, even under strongstress conditions as 40° C.±2° C./75% RH±5% for 5 weeks.

PSMA binding data for the 5 weeks' time point at 40° C.±2° C./75% RH±5%showed a strong increase in the EC₅₀ for 10 mg/mL samples in buffer 1and 3. The increase in the EC₅₀ measured in the PSMA binding assaycorrelated with the slight increase in the relative HMW species amountobserved in the SE-HPLC analysis from 0.3 and 0.2% to 0.6 and 0.7%,respectively.

The results from the stress tests (freeze/thawing (five F/T cycles),photo-, agitation- and oxidation stress) showed a different ranking thanthe temperature stability study. In the temperature stability study, 10mg/mL samples in buffer 3 showed the highest amounts of results outsidetheir predefined analytical threshold, whereas the stress stabilitystudy revealed increased results outside their predefined analyticalthreshold for 0.5 mg/mL samples in buffer 1 compared to the otherconditions.

LC-MS analysis revealed that the addition of methionine (present inbuffer 2 and buffer 4) to prevent oxidation of the product waseffective.

In conclusion, LV1050-Fc x 5C8var1-Fc was most stable when formulated inbuffer 2 and buffer 4. An overview of the sum of results outside theirpredefined analytical threshold for temperature stability, stressstability and overall stability (temperature plus stress) is shown inTable 8.

For final formulation, 10 mM Histidine+280 mM Sucrose+0.02% Polysorbate80, pH 6.0+1 mM Methionine was chosen, at a protein concentrationbetween 0.5 and 10 mg/mL.

TABLE 8 Sum of results outside their predefined analytical threshold fortemperature stability, stress stability and overall stability(temperature plus stress). Buffer Buffer 1 Buffer 2 Buffer 3 Buffer 4Protein conc. 0.5 10 0.5 10 0.5 10 0.5 10 (mg/mL) Temperature 0 2 0 0 16 0 0 stability Stress 11 5 5 4 5 5 5 4 stability Overall 11 7 5 4 6 115 4 stability

1. A multispecific antibody comprising a first antigen-binding region capable of binding human PSMA and a second antigen-binding region capable of binding a human Vγ9Vβ2 T cell receptor.
 2. The multispecific antibody according to claim 1, wherein the multispecific antibody is a bispecific antibody.
 3. The multispecific antibody according to claim 1, wherein the first antigen-binding region is a single-domain antibody.
 4. The multispecific antibody according to claim 1, wherein the second antigen-binding region is a single-domain antibody.
 5. The multispecific antibody according to claim 1, wherein the multispecific antibody competes for binding to human PSMA with an antibody having the sequence set forth in SEQ ID NO:2.
 6. The multispecific antibody according to a claim 1, wherein the first antigen-binding region comprises the VH CDR1 sequence set forth in SEQ ID NO:14, the VH CDR2 sequence set forth in SEQ ID NO:15 and the VH CDR3 sequence set forth in SEQ ID NO:16.
 7. The multispecific antibody according to claim 1, wherein the first antigen-binding region is humanized, and wherein the first antigen-binding region comprises the sequence set forth in SEQ ID NO:2, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:2.
 8. The multispecific antibody according to claim 1, wherein the multispecific antibody is able to activate human Vγ9Vβ2 T cells.
 9. The multispecific antibody according to claim 1, wherein the multispecific antibody is capable of binding to human Vβ2.
 10. The multispecific antibody according to claim 1, wherein the multispecific antibody competes for binding to human Vβ2 with an antibody having the sequence set forth in SEQ ID NO:5 or competes for binding to human Vβ2 with an antibody having the sequence set forth in SEQ ID NO:20.
 11. The multispecific antibody according to claim 1, wherein the multispecific antibody binds the same epitope on human Vβ2 as an antibody having the sequence set forth in SEQ ID NO:5 or binds the same epitope on human Vβ2 as an antibody having the sequence set forth in SEQ ID NO:20.
 12. The multispecific antibody according to claim 1, wherein the second antigen-binding region comprises the VH CDR1 sequence set forth in SEQ ID NO:17, the VH CDR2 sequence set forth in SEQ ID NO:18 and the VH CDR3 sequence set forth in SEQ ID NO:19 or comprises the VH CDR1 sequence set forth in SEQ ID NO:21, the VH CDR2 sequence set forth in SEQ ID NO:22 and the VH CDR3 sequence set forth in SEQ ID NO:23.
 13. The multispecific antibody according to claim 1, wherein the second antigen-binding region is humanized, and wherein the second antigen-binding region comprises the sequence set forth in SEQ ID NO:5, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:5.
 14. The multispecific antibody according to claim 1, wherein the first antigen-binding region comprises the VH CDR1 sequence set forth in SEQ ID NO:14, the VH CDR2 sequence set forth in SEQ ID NO:15 and the VH CDR3 sequence set forth in SEQ ID NO:16 and wherein the second antigen-binding region comprises the VH CDR1 sequence set forth in SEQ ID NO:15, the VH CDR2 sequence set forth in SEQ ID NO:18 and the VH CDR3 sequence set forth in SEQ ID NO:19.
 15. The multispecific antibody according to claim 1, wherein the multispecific antibody is capable of binding to human Vg9.
 16. The multispecific antibody according to claim 1, wherein the multispecific antibody is capable of mediating killing of PSMA-expressing cells by Vγ9Vβ2 T cells.
 17. The multispecific antibody according to claim 1, wherein the multispecific antibody is capable of mediating killing of human PSMA-expressing cells from a prostate cancer patient.
 18. The multispecific antibody according to claim 1, wherein the first antigen-binding region and second antigen-binding region are covalently linked via a peptide linker.
 19. The multispecific antibody according to claim 18, wherein the peptide linker comprises the sequence set forth in SEQ ID NO:6.
 20. The multispecific antibody according to claim 1, wherein the first antigen-binding region capable of binding human PSMA is located N-terminally of the second antigen-binding region capable of binding a human Vγ9Vβ2 T cell receptor.
 21. The multispecific antibody according to claim 1, wherein the multispecific antibody further comprises a half-life extension domain.
 22. The multispecific antibody according to claim 20, wherein the multispecific antibody has a terminal half-life that is longer than about 168 hours when administered to a human subject.
 23. The multispecific antibody according to claim 1, wherein the multispecific antibody comprises an Fc region.
 24. The multispecific antibody according to claim 23, wherein the Fc region is a heterodimer comprising two Fc polypeptides, wherein the first antigen-binding region is fused to the first Fc polypeptide and the second antigen-binding region is fused to the second Fc polypeptide and wherein the first and second Fc polypeptides comprise asymmetric amino acid mutations that favor the formation of heterodimers over the formation of homodimers.
 25. The multispecific antibody according to claim 24, wherein the CH3 regions of the Fc polypeptides comprise said asymmetric amino acid mutations, wherein the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A and Y407V substitutions, or vice versa, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.
 26. The multispecific antibody according to claim 24, wherein the cysteine residues at position 220 in the first and second Fc polypeptides have been deleted or substituted, and wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.
 27. The multispecific antibody according claim 24, wherein the first and second Fc polypeptides further comprise a mutation at position 234 and/or 235, wherein the first and second Fc polypeptide comprise an L234F and an L235E substitution, and wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.
 28. The multispecific antibody according to claim 24, wherein the first antigen-binding region comprises the sequence set forth in SEQ ID NO:2, the second antigen-binding region comprises the sequence set forth in SEQ ID NO:5 and wherein the first Fc polypeptide comprises the sequence set forth in SEQ ID NO:12 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:13, or the first Fc polypeptide comprises the sequence set forth in SEQ ID NO:12 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:13.
 29. A pharmaceutical composition comprising a multispecific antibody according to claim 1, and a pharmaceutically-acceptable excipient.
 30. The pharmaceutical composition according to claim 29, wherein the pharmaceutical composition comprises a buffer, sucrose, polysorbate 80 and methionine, and wherein the pH of the composition is between 5.4 and 7.4.
 31. The A medicament comprising a multispecific antibody according to claim
 1. 32. The A method of treating cancer comprising administration of the multispecific antibody according to claim
 1. 33. A method of treating prostate cancer comprising administration of the multispecific antibody according to claim
 1. 34. The A method of treating cancers in which PSMA is expressed on the tumor neo-vasculature or tumor-associated endothelial cells of primary or metastatic tumors comprising cancers selected from colorectal cancer, lung cancer, breast cancer, endometrial and ovarian cancer, gastric cancer, renal cell cancer, urothelial cancer, hepatocellular cancer, oral squamous cancer, thyroid tumors and glioblastomas comprising administration of the multispecific antibody according to claim
 1. 35. A method of treating an adenoid cystic carcinoma of the head and neck comprising administration of the multispecific antibody according to claim
 1. 36. A method of treating a disease comprising administration of a multispecific antibody according to claim
 1. 37. The method according to claim 36, wherein the disease is cancer.
 38. A nucleic acid construct encoding the multispecific antibody according to claim
 1. 39. An expression vector comprising a nucleic acid construct according to claim
 38. 40. A host cell comprising a nucleic acid construct according to claim
 38. 41. A method for production of the multispecific antibody as defined in claim 1 comprising (co-)expression of one or more nucleic acid constructs encoding the multispecific antibody in a suitable host cell, followed by purification of the produced recombinant antibody.
 42. The multispecific antibody according to claim 1, wherein the multispecific antibody binds the same epitope on human PSMA as an antibody having the sequence set forth in SEQ ID NO:2.
 43. The multispecific antibody according to claim 16, wherein the PSMA-expressing cells comprise LNCaP cells.
 44. The pharmaceutical composition according to claim 30, wherein the pH of the composition is between 5.4 and 6.1.
 45. A host cell comprising an expression vector according to claim
 39. 46. The host cell according to claim 40, wherein the host cell is a mammalian host cell.
 47. The host cell according to claim 46, wherein the mammalian host cell is a CHO cell. 